Compositions and methods for treating cancer

ABSTRACT

The present invention provides, in part, compositions and methods for treating cancer using a combination of C6-ceramide and other anti-cancer agents.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/110,318, which is a 35 U.S.C. § 371 U.S. national stage entry ofInternational Application PCT/US2012/032143 having an internationalfiling date of Apr. 4, 2012, which claims the benefit of priority toU.S. Provisional Application No. 61/472,266, filed on Apr. 6, 2011, allof which are hereby incorporated by reference for all purposes as iffully set forth herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 17, 2012, isnamed WAR00125.txt and is 5,303 bytes in size.

BACKGROUND OF THE INVENTION

Membrane sphingolipids have been shown to be biologically active andexert numerous regulatory effects on cellular functions includingmodulating cell growth and differentiation. Ceramides, found in highconcentrations within the cell membrane, are a family of lipid moleculescomposed of sphingosine and a fatty acid which function as structuralelements, as well as signaling molecules. Studies have demonstratedimportant relationships between ceramide production and apoptosis intumor cells and suggest that processes which enhance intracellularceramide accumulation may provide in favorable proapoptotic effectsduring cancer chemotherapy (Bose et al. (1995) Cell 82:405-414; Mathiaset al. (1998) Biochem. J. 335 (Pt 3): 465-480). Cell permeable shortchain ceramides (C2- or C6-ceramide) have shown activity relevant totherapeutically treating cancer indications. For example, such ceramideforms have an anti-cancer effect on many cancer cell lines (reviewed inRadin (2003) Biochem. J. 371 (Pt 2): 243-256), including melanoma andsoft tissue sarcoma (Auzenne et al. (1998) Melanoma Res. 8:227-239),Jurkat leukemia (Myrick et al. (1999) Leuk. Res. 23:569-578), and headand neck squamous cancer (Mehta et al. (2000) Cancer Chemother.Pharmacol. 46:85-92) cell lines. Ceramides C2, C6 and their analogueshave also been shown to induce cell cycle arrest in a variety of tumortypes (reviewed in Mathias et al. (1998) Biochem J. 335 (Pt 3):465-480). Generation of endogenous ceramide has been shown to mediateapoptosis induced by a variety of anti-cancer drugs (reviewed in Mathiaset al. (1998) Biochem. J. 335 (Pt 3): 465-480) including daunorubicin(Reddy et al. (2000) J. Immunol. 164:1355-1363), doxorubicin (Lucci etal. (1999) Cancer 86:300-311), ara-C (Strum et al. (1994) J. Biol. Chem.269:15493-15497), suramin (Safavy et al. (2003) Bioconjug. Chem.14:302-310), and paclitaxel (Charles et al. (2001) Cancer Chemother.Pharmacol. 47:444-450). Despite these observations, however, themolecular mechanisms underlying the therapeutically beneficial effectsof ceramide, particularly cell permeable ceramides such as C6-ceramide,are unknown. This lack of understanding has hindered the development ofcompositions and methods containing ceramide (e.g., C6-ceramide) incombination with other agents that enhance the specific anti-cancerpathways affected by ceramide and/or overcome the pro-survival sideeffects of many anti-cancer therapeutics currently used in the clinic.Accordingly, there is a great need in the art to better understand themolecular mechanisms underlying the anti-cancer effect of ceramide andits derivatives.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatthe use of ceramide (e.g., C6-ceramide), in combination with one or moreanti-cancer agents, e.g., a chemotherapeutic agent or EGFR inhibitor,for the treatment of certain cancers (e.g., cancers characterized byhyperactive KRAS mutant polypeptides and/or pancreatic or colorectalcancer).

In one aspect, the present invention provides a method for treatingcancer, wherein the method comprises contacting a cancer cell having anactivating KRAS mutation with (a) an effective amount of C6-ceramide,and (b) an effective amount of at least one anti-cancer agent, therebytreating cancer.

In another aspect, the present invention provides a method for treatingcancer, wherein the method comprises contacting a cancer cell with (a)an effective amount of C6-ceramide; (b) an effective amount of ananti-cancer agent; and (c) an effective amount of an agent selected fromthe group consisting of an enhancer of the AMPK signaling pathway, aninhibitor of the PI3K/AKT/mTORC1 signaling pathway, and an inhibitor ofthe MEK/ERK signaling pathway, thereby treating cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show that C6-ceramide dramatically enhances taxol inducedcell death in L3.6 pancreatic cancer cells in vitro. L3.6 pancreaticcancer cells, cultured either in the basic DMEM medium or with 10% FBS,were treated with the indicated doses of taxol in the presence orabsence of C6-ceramide (10 μg/ml) for 48 hours and cell viability wasdetected by MTT assay (FIGS. 1A-1B). L3.6 cells, cultured either inbasic DMEM medium or with 10% FBS, were treated with the indicated doseof C6-ceramide in the presence or absence of taxol (1.5 μg/ml) for 48hours and cell viability was detected by MTT assay (FIGS. 1C-1D).Mitomycin C (10 μg/ml) was always present in the media to prevent cellproliferation from occurring. The data represents the mean±SD of atleast triplicate experiments. *P<0.05 versus group without C6-ceramidepresents (FIGS. 1A-1B). **P<0.05 versus group without taxol presents(FIGS. 1C-1D).

FIGS. 2A-2E show that C6-ceramide and paclitaxel cause synergisticanti-tumor effects in vivo, SCID/Beige/Taconic nude male mice, 22-25 g,6-8 weeks old were ear tagged and randomized into 4 different groups(Control, ceramide, paclitaxel, ceramide+paclitaxel) of 5 mice eachprior to inoculation subcutaneously (s.c.) with 2×10⁶ L3.6 cells in avolume 0.1 ml into the internal surface of the right thigh. Treatmentwas started 4 days after L3.6 cell injection with thrice weekly (3×/wk)intraperitoneal (i.p.) injections of paclitaxel 3.0 mg/ml with orwithout C6-ceramide (10 mg/ml) for 2 weeks. Mouse survival (FIGS. 2A and2C), tumor volume (in cm³; FIG. 2B), mouse body weight (in grams) wererecorded (FIG. 2E). The average rate of tumor development was calculatedby total tumor volume dividing by total number of days monitored (FIG.2C). The experiments were repeated at least three times and similarresults were observed.

FIGS. 3A-3H show synergistic anti-tumor effects of C6-ceramide andgemcitabine in vivo and in vitro. SCID/Beige/Taconic nude male mice(22-25 g; 6-8 weeks old) were ear tagged and randomized into 4 differentgroups (Control, Ceramide, gemcitabine, Ceramide+gemcitabine) combining5 mice each prior to inoculation subcutaneous (s.c.) with 2×10⁶ L3.6cells in a 0.1 ml volume into the internal surface of the right thigh.Treatment was started 4 days after L3.6 cell injection with thriceweekly (3×/wk) intraperitoneal (i.p.) injections of gemcitabine (twodoses: 5.0 and 10.0 mg/ml) with or without C6-ceramide (10 mg/ml) for 2weeks. Mouse survival (FIGS. 3A, 3B, and 3E) and tumor volume (in cm³;FIGS. 3C and 3D) were recorded (FIG. 3E). Pancreatic cancer cell lines,including L3.6 cells (FIG. 3F), Panc-1 cells (FIG. 3G) and MIA-PaCa2cells (MIA, FIG. 3H) were cultured in basic DMEM medium and were treatedwith gemcitabine (Gem, 1 μg/ml). Mitomycin C was always present in themedia to prevent cell proliferation from occurring. The data representthe mean±SD of at least triplicate experiments. *P<0.05 versus groupwithout C6-ceramide presents. The experiments were repeated at leastthree times and similar results were observed.

FIGS. 4A-4F show that PI3K/AKT/mTOR inhibition and AMPK activationenhance taxol induced cancer cell death.

FIG. 5A-5F show that C6-ceramide plus gemcitabine or taxol causesinactivation of AKT/mTORC1 and ERK in vitro. Pancreatic cancer celllines L3.6 cells were left untreated or treated with gemcitabine (Gem, 1μg/ml) or taxol (1.5 μg/ml) in the presence or absence of C6-ceramide(10 μg/ml) for 2, 4 and 6 hours, AKT/mTORC1 and ERK activation weredetected by Western Blot using indicated antibodies (FIGS. 5A and 5C).p-AKT (S473) and p-ERK at 4 hour time intervals for each treatmentquantified (FIGS. 5B and 5D). L3.6 cells were pretreated with variousinhibitors including PI3K/AKT inhibitor LY 294002 (LY, 1 μM), mTORC1inhibitor rapamycin (100 nM), MEK/ERK inhibitor U0126 (1 μg/ml) orgemcitabine (1 μg/ml) for 48 hours, cell death was detected 48 hourslater (FIG. 5E). The effects of the various inhibitors described aboveon AKT/mTORC1 and ERK activation were also detected by Western Blotsusing commercial available antibodies (FIG. 5F). *P<0.05 versus groupwithout C6-ceramide presents. The experiments were repeated at leastthree times and similar results were observed.

FIG. 6 shows a schematic outline of biological pathways affected byC6-ceramide.

FIG. 7 shows that ceramide sensitizes KRAS mutated pancreatic cancercells to cetuximab.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of ceramide (e.g.,C6-ceramide), in combination with one or more anti-cancer agents, e.g.,a chemotherapeutic agent, for the treatment of cancer. The presentinvention also relates to compositions and methods for treating certaincancers (e.g., cancers characterized by hyperactive KRAS mutantpolypeptides and/or pancreatic or colorectal cancer), wherein cancercells are contacted with an effective amount of ceramide (e.g.,C6-ceramide) and an effective amount of at least one (i.e., one or more)anti-cancer agents (e.g., a chemotherapeutic agent, an enhancer of theAMPK signaling pathway, an inhibitor of the PI3K/AKT/mTORC1 signalingpathway, an inhibitor of the MEK/ERK signaling pathway, and combinationsthereof).

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

I. Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “activating KRAS mutation” refers to a mutationin a KRAS polypeptide that causes enhanced KRAS activity relative to thecontrol wild-type KRAS polypeptide without the mutation. In oneembodiment, the activating KRAS mutation is selected from the groupconsisting of G12C; G12A; G12D; G12R; G12S; G12V; G13C; G13D of thehuman KRAS polypeptide. The term “altered activity” refers to anactivity of a molecule (e.g., a polypeptide) which is increased ordecreased in a defined state (e.g., in a mutated or diseased stateand/or sample), as compared to the activity of the biomarker in acontrol state (e.g., in a wild type or normal, control state and/orsample). Altered activity may be the result of, for example, alteredexpression of the biomarker, altered protein level of the biomarker,altered structure of the biomarker, or, e.g., an altered interactionwith other proteins involved in the same or different pathway as thebiomarker or altered interaction with transcriptional activators orinhibitors, or altered methylation status. Such altered activity can beat least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,250%, 300%, 350%, 400%, 450%, 500%, 1000%, or more modulated (e.g.,upregulated or downregulated).

As used herein, the term “anti-cancer response” to therapy relates toany response of the cancer to therapy, preferably to a change in tumormass and/or volume after initiation of neoadjuvant or adjuvantchemotherapy. Hyperproliferative disorder response may be assessed in aneoadjuvant or adjuvant situation where the size of a tumor aftersystemic intervention can be compared to the initial size and dimensionsas measured by CT, PET, mammogram, ultrasound or palpation. Response mayalso be assessed by caliper measurement or pathological examination ofthe tumor after biopsy or surgical resection. Response may be recordedin a quantitative fashion like percentage change in tumor volume or in aqualitative fashion like “pathological complete response” (pCR),“clinical complete remission” (cCR), “clinical partial remission” (cPR),“clinical stable disease” (cSD), “clinical progressive disease” (cPD) orother qualitative criteria. Assessment of hyperproliferative disorderresponse may be done early after the onset of neoadjuvant or adjuvanttherapy, e.g., after a few hours, days, weeks or preferably after a fewmonths. A typical endpoint for response assessment is upon terminationof neoadjuvant chemotherapy or upon surgical removal of residual tumorcells and/or the tumor bed. This is typically three months afterinitiation of neoadjuvant therapy.

As used herein, the term “body fluid” refers to fluids that are excretedor secreted from the body as well as fluid that are normally not (e.g.amniotic fluid, aqueous humor, bile, blood and blood plasma,cerebrospinal fluid, cerumen and earwax, cowper's fluid orpre-ejaculatory fluid, chyle, chyme, stool, female ejaculate,interstitial fluid, intracellular fluid, lymph, menses, breast milk,mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovialfluid, tears, urine, vaginal lubrication, vitreous humor, vomit).

As used herein, the term “cancer” is intended to encompass a tumor,including both in vitro and in vivo tumors that form in any organ orbody part of the subject. Examples of the types of tumors intended to beencompassed by the present invention include those tumors associatedwith breast cancer, skin cancer, bone cancer, prostate cancer, livercancer, lung cancer, brain cancer, cancer of the larynx, gallbladder,pancreas, rectum, parathyroid, thyroid, adrenal, neural tissue, head andneck, colon, stomach, bronchi, kidneys. Specifically, the tumors whosegrowth rate is inhibited by the present invention include basal cellcarcinoma, squamous cell carcinoma of both ulcerating and papillarytype, metastatic skin carcinoma, osteo sarcoma, Ewing's sarcoma,veticulum cell sarcoma, myeloma, giant cell tumor, small-cell lungtumor, gallstones, islet cell tumor, primary brain tumor, acute andchronic lymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,hyperplasia, medullary carcinoma, pheochromocytoma, mucosal neuromas,intestinal ganglloneuromas, hyperplastic corneal nerve tumor, marfanoidhabitus tumor, Wilm's tumor, seminoma, ovarian tumor, leiomyomatertumor, cervical dysplasia and in situ carcinoma, neuroblastoma,retinoblastoma, soft tissue sarcoma, malignant carcinoid, topical skinlesion, mycosis fungoide, rhabdomyosarcoma, Kaposi's sarcoma, osteogenicand other sarcoma, malignant hypercalcemia, renal cell tumor,polycythermia vera, adenocarcinoma, glioblastoma multiforma, leukemias,lymphomas, malignant melanomas, epidermoid carcinomas, and othercarcinomas and sarcomas.

In one embodiment, the cancer is pancreatic cancer. The term “pancreaticcancer” as used herein, includes adenomas, adenocarcinomas, gastrinomas,somatostatinomas, insulinomas and glucagonomas of the pancreas. As usedherein, the term “adenocarcinoma” is carcinoma that develops in thelining or inner surface of an organ and is derived from glandular tissueor in which the tumor cells form recognizable glandular structures. Asused interchangeably herein, the terms, “pancreatic adenocarcinoma,” or“pancreatic ductal adenocarcinoma” is an adenocarcinoma of the pancreas.In one embodiment, pancreatic adenocarcinomas arise from the progressionof lesions that occur in the pancreatic ducts (pancreaticintraepithelial neoplasia, referred to herein as “PanIN”). Pancreaticcancer is a malignant growth of the pancreas that mainly occurs in thecells of the pancreatic ducts. This disease is the ninth most commonform of cancer, yet it is the fourth and fifth leading cause of cancerdeaths in men and women, respectively. Cancer of the pancreas is almostalways fatal, with a five-year survival rate that is less than 3%. Themost common symptoms of pancreatic cancer include jaundice, abdominalpain, and weight loss, which, together with other presenting factors,are nonspecific in nature. Thus, diagnosing pancreatic cancer at anearly stage of tumor growth is often difficult and requires extensivediagnostic work-up, often times including exploratory surgery.Endoscopic ultrasonography and computed tomography are the bestnoninvasive means available today for diagnosis of pancreatic cancer.However, reliable detection of small tumors, as well as differentiationof pancreatic cancer from focal pancreatitis, is difficult. The vastmajority of patients with pancreatic cancer are presently diagnosed at alate stage when the tumor has already extended outside of the capsule toinvade surrounding organs and/or has metastasized extensively (Gold etal. (2001) Crit. Rev. Oncology/Hematology, 39:147-54). Late detection ofthe disease is common, and early pancreatic cancer diagnosis is rare inthe clinical setting. This is significant, since late detection ofpancreatic cancer results in low survival rate. Current treatmentprocedures available for pancreatic cancer have not led to a cure, or toa substantially improved survival time. Surgical resection has been theonly modality that offers a chance at survival. However, due to a largetumor burden, only 10% to 25% of patients are candidates for “curativeresection.” For those patients undergoing a surgical treatment, thefive-year survival rate is still poor, averaging only about 10%. A“non-endocrine pancreatic cancer” generally refers to cancers arisingfrom the exocrine pancreatic glands. The term excludes pancreaticinsulinomas and includes pancreatic carcinoma, pancreaticadenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma andgiant cell carcinoma and precursor lesions such as pancreaticintra-epithelial neoplasia (PanIN), mucinous cyst neoplasms (MCN) andintrapancreatic mucinous neoplasms (IPMN), which are neoplastic but notyet malignant. The terms “pancreatic cancer” and “non-endocrinepancreatic cancer” are used interchangeably herein.

In another embodiment, the cancer is colorectal cancer. Colon cancer islocated in the large intestine, while rectal cancer is in the rectum.The difference between these two cancers is the location in the largeintestine where the cancer occurs. Therefore, the term colorectal canceris often used to refer to cancer in both locations. Colorectal cancer isthird most common leading causes of cancer death in the United States.According to the “American Cancer Society Colorectal Cancer Facts andFigures 2011-2013” (Atlanta, American Cancer Society, 2011), in 2001,the incidence rates of colorectal cancer per 100,000 are about 57.2among male, and about 42.5 among female. In comparison, the mortalityrates are 21.2 among male, and 14.9 among female per 100,000. As awhole, there will be about 141,000 new cases and 49,000 deaths in 2011.The common stages of colorectal cancer includes: Stage 0: when cancer isonly on the innermost layer of the intestine; Stage I: when cancer is inthe inner layer of the colon; Stage II: when cancer has spread throughthe muscle wall of the colon; Stage III: when cancer has spread to thelymph nodes; and Stage IV: when cancer has spread to other organs. Themost effective approach to treat colorectal cancer is early detectionbefore symptoms develop by undergoing periodic colonoscopy orsigmoidoscopy when a person is 50 years or older, or has either a familyhistory or personal history of colon cancer. The treatment options ofcolorectal cancer are surgery, radiation therapy and chemotherapy.5-Fluorouracil, oxaliplatin and irinoteccan are commonly usedchemotherapeutic agents for colorectal cancer.

As used herein, the term “cancer cell” is intended to include tumorcells, and refers to cells that divide at an abnormal (increased) rate.Cancer cells include, but are not limited to, carcinomas, such assquamous cell carcinoma, basal cell carcinoma, sweat gland carcinoma,sebaccous gland carcinoma, adenocarcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma,undifferentiated carcinoma, bronchogenic carcinoma, melanoma, renal cellcarcinoma, hepatoma-liver cell carcinoma, bile duct carcinoma,chlolangiocarcinoma, papillary carcinoma, transitional cell carcinoma,choriocarcinoma, semonoma, embryonal carcinoma, mammary carcinomas,gastrointestinal carcinoma, colonic carcinomas, bladder carcinoma,prostate carcinoma, and squamous cell carcinoma of the neck and headregion; sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, synoviosarcoma andmesotheliosarcoma; leukemias and lymphomas such as granulocyticleukemia, monocytic leukemia, lymphocytic leukemia, malignant lymphoma,plasmocytoma, reticulum cell sarcoma, or Hodgkin's disease; and tumorsof the nervous system including glioma, meningioma, medulloblastoma,schwannoma or epidymoma.

As used herein “contacting cancer cells” is defined as exposing thecancer cells to one or more combinations of agents described herein. Inone embodiment, such combinations can be administered to cancer cells,directly or indirectly, using local, regional or systemic means.

As used herein a “cremophore” is a solvent that permits solubilizationof a drug or compound. Various cremophores are well known to one ofskill in the art, including but not limited to oil-based solvents.

As used herein “decreasing the size of a tumor” is defined as areduction in the size of a tumor. Such an effect can be accomplished byreducing the number of proliferating tumor cells in the tumor (e.g., byreducing cell division of the tumor cells) and/or by inducingcytotoxicity or cell death (apoptosis) of existing tumor cells.Accordingly, tumor growth is arrested or prevented.

As used herein, the term “EGFR inhibitor” refers to an inhibitor of theepidermal growth factor receptor (EGFR). In one embodiment, the EGFRinhibitor is an antibody such as Erbitux™ (cetuximab, Imclone SystemsInc.) and ABX-EGF (panitumumab, Abgenix, Inc.). In another embodimentthe EGFR inhibitor is a small molecule that competes with ATP such asTarceva™ (erlotinib, OSI Pharmaceuticals), Iressa™ (gefitinib,Astra-Zeneca), tyrphostins described by Dvir et al. (1991) JCB113:857-865; tricyclic pyrimidine compounds disclosed in U.S. Pat. No.5,679,683; compound6-(2,6-dichlorophenyl)-2-(4-(2-diethylaminoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3-d)pyrimidin-7-one(known as PD166285) disclosed in Panek et al. (1997) J. Pharm. Exp.Therap. 283:1433-1444).

As used herein “increasing apoptosis” is defined as an increase in therate of programmed cell death, i.e. more cells are induced into thedeath process as compared to exposure (contact with) either gemcitabinealone or the ceramide alone. Increasing apoptosis also includes theinhibition of cell division which results in a decrease in the totalnumber of viable cancer cells.

As used herein, the term “inhibiting cancer” or “inhibiting cancer cellgrowth” is intended to include the inhibition of undesirable orinappropriate cell growth. The inhibition is intended to includeinhibition of proliferation including rapid proliferation. The term“inhibiting cancer cell growth” is also intended to encompass inhibitingtumor growth which includes the prevention of the growth of a tumor in asubject or a reduction in the growth of a pre-existing tumor in asubject. The inhibition also can be the inhibition of the metastasis ofa tumor from one site to another. A cancer is “inhibited” if at leastone symptom of the cancer is alleviated, terminated, slowed, orprevented. As used herein, cancer is also “inhibited” if recurrence ormetastasis of the cancer is reduced, slowed, delayed, or prevented.

The term “modulate” includes downregulation and upregulation. The term“downregulate,” “decrease,” “reduce,” “inhibit,” and the like are allused herein generally to mean a decrease by a statistically significantamount. The term “upregulate,” “increase,” “enhance,” and the like areall used herein generally to mean an increase by a statisticallysignificant amount. For example, an increase or a decrease can be by atleast about 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 450%, 500%, 1000%, or more or any range in between 10-1000%inclusive as compared to a control. In some embodiments, the control canbe a change in a cancer cell state such as cancer cell proliferation inthe presence versus the absence of treatment. In another embodiment, thecontrol can be activity of a wild type polypeptide of interest. An“overactivity” or “significantly higher level of activity” refers to anactivity level of a molecule or test sample that is greater than thestandard error of the assay employed to assess the activity, and ispreferably at least twice, and more preferably three, four, five or tenor more times the activity relative to a reference or control sample andpreferably, the average activity in several control samples. the term“underactivity” refers to the opposite of “overactivity.”

A cancer cell is “resistant” to a therapeutic agent if its rate ofgrowth is not inhibited, or inhibited to a very low degree, as a resultof contact with the therapeutic agent when compared to its growth in theabsence of contact with the therapeutic agent. The quality of beingresistant to a therapeutic agent is a highly variable one, withdifferent cancer cells exhibiting different levels of “resistance” to agiven therapeutic agent under different conditions.

A cancer cell is “sensitive” to a therapeutic agent if its rate ofgrowth is inhibited as a result of contact with a therapeutic agent,compared to its growth in the absence of contact with the therapeuticagent. The quality of being sensitive to a therapeutic agent is avariable one, with different cancer cells exhibiting different levels of“sensitivity” to a given therapeutic agent, under different conditions.

Determination of whether a patient is “sensitive” or “resistant” to atherapeutic agent and/or protocol can be readily made by the physician(the “attending clinician”), as one skilled in the art, by the use ofknown techniques. For example, a number of factors are considered by theattending clinician, including, but not limited to: the specific cancerinvolved; pharmacodynamic characteristics of the particular therapeuticagent; the size, age, and general health of the patient; the degree ofor involvement or the severity of the cancer; the particular compoundadministered; the mode of administration; and other relevantcircumstances.

As used herein, the term “survival” includes all of the following:survival until mortality, also known as overall survival (wherein saidmortality may be either irrespective of cause or tumor related);“recurrence-free survival” (wherein the term recurrence shall includeboth localized and distant recurrence); metastasis free survival;disease free survival (wherein the term disease shall include cancer anddiseases associated therewith). The length of said survival may becalculated by reference to a defined start point (e.g., time ofdiagnosis or start of treatment) and end point (e.g., death, recurrenceor metastasis). In addition, criteria for efficacy of treatment can beexpanded to include response to chemotherapy, probability of survival,probability of metastasis within a given time period, and probability oftumor recurrence.

As used herein, the term “subject” shall mean any animal including,without limitation, a human, a mouse, a rat, a rabbit, a non-humanprimate, or any other mammal. In one embodiment, the subject is aprimate. In another embodiment, the subject is a human.

As used herein, the term “synergistic” refers to a combination oftherapeutic agents described herein, which, when taken together, is moreeffective than the additive effects of the individual therapies. Asynergistic effect of a combination of therapies (e.g., a combination oftherapeutic agents) permits the use of lower dosages of one or more ofthe therapeutic agent(s) and/or less frequent administration of theagent(s) to a subject with a disease or disorder, e.g., a proliferativedisorder. The ability to utilize lower the dosage of one or moretherapeutic agent and/or to administer the therapeutic agent lessfrequently reduces the toxicity associated with the administration ofthe agent to a subject without reducing the efficacy of the therapy inthe treatment of a disease or disorder. In addition, a synergisticeffect can result in improved efficacy of agents in the prevention,management or treatment of a disease or disorder, e.g. a proliferativedisorder. Finally, a synergistic effect of a combination of therapiesmay avoid or reduce adverse or unwanted side effects associated with theuse of either therapeutic agent alone. As used herein, the term “incombination” refers to the use of more than one therapeutic agent. Theuse of the term “in combination” does not restrict the order in whichthe therapeutic agents are administered to a subject with a disease ordisorder, e.g., a proliferative disorder. A first therapeutic agent,such as a compound described herein, can be administered prior to (e.g.,5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours,6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of asecond therapeutic agent, such as an anti-cancer agent, to a subjectwith a disease or disorder, e.g. a proliferative disorder, such ascancer.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or homologous with all or a portion of amature mRNA made by transcription of a marker of the invention andnormal post-transcriptional processing (e.g. splicing), if any, of theRNA transcript, and reverse transcription of the RNA transcript.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid and the amino acidsequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNA codingfor a fusions protein or polypeptide of the invention (or any portionthereof) can be used to derive the fusion protein or polypeptide aminoacid sequence, using the genetic code to translate the DNA or RNA intoan amino acid sequence. Likewise, for fusion protein or polypeptideamino acid sequence, corresponding nucleotide sequences that can encodethe fusion protein or polypeptide can be deduced from the genetic code(which, because of its redundancy, will produce multiple nucleic acidsequences for any given amino acid sequence). Thus, description and/ordisclosure herein of a nucleotide sequence which encodes a fusionprotein or polypeptide should be considered to also include descriptionand/or disclosure of the amino acid sequence encoded by the nucleotidesequence. Similarly, description and/or disclosure of a fusion proteinor polypeptide amino acid sequence herein should be considered to alsoinclude description and/or disclosure of all possible nucleotidesequences that can encode the amino acid sequence.

II. Therapeutic Agents and Pharmaceutical Compositions

The methods of the present invention use combinations of therapeuticagents in a pharmaceutical composition as described below.

A. Ceramide

The term “ceramide” generally refers to any N-acylsphingosine. Ceramidesinclude sphingolipids in which the sphingosine is acylated with a fattyacid acyl CoA derivative to form an N-acylsphingosine. Ceramide may beeither naturally occurring or chemically synthesized. Preferably, thecarbon chain length is less than 18 carbons. Examples includeC6-ceramide (N-hexanoyl-D-sphingosine), C2-ceramide(N-acetyl-D-sphingosine), C8-ceramide (N-octyl-D-sphingosine) andC16-ceramide (N-palmitoyl-D-sphingosine). Other ceramides are known topersons having ordinary skill in the art. In certain embodiments of theabove-described methods and composition, the ceramide may be aC2-ceramide, C6-ceramide, C8-ceramide, C16-ceramide, or a higher orderof ceramide. In one embodiment, the ceramide is C6-ceramide. For eachembodiment of the present invention described herein relating toC6-ceramide, each of the other orders of ceramide known to the skilledartisan are also contemplated mutatis mutandis. Ceramide, which isnormally lipid soluble, can be made water soluble according towell-known methods in order to enable contact with cancer cells (e.g.,in a subject). Ceramide may be solubilized initially in alcohol and thensubsequently diluted in saline or a cremophore.

The amount of ceramide is from about 1.0 mg/kg-about 10.0 mg/kg everytwo weeks. In a further embodiment, the amount of ceramide is about 10.0mg/kg every two weeks. In a further embodiment, the amount of ceramideis about 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10.0 mg/kg, 11.0 mg/kg, 13.0 mg/kg, 14.0mg/kg or 15.0 mg/kg every two weeks.

For each embodiment described herein, the ratio of ceramide to othertherapeutic agent composition of a therapeutic combination describedherein can be 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5:1,5.5:1, 6:1, 6.5:1, 7:1, 7.5:1 or greater or any range in between.

B. Therapeutic Agents

As used herein, the term “anti-cancer agent” and “therapeutic agent” isdefined broadly as anything that cancer cells, including tumor cells,may be exposed to in a therapeutic protocol for the purpose ofinhibiting their growth or kill the cells. In one embodiments, suchagents can be used according to the methods described herein either inconjunction with ceramide (e.g., C6-ceramide), in conjunction with eachother (e.g., LY294002 plus gemcitabine, taxol plus U0126, taxol plusgemcitabine, etc.), or in any combination thereof. In the context of thepresent invention, such agents include, but are not limited to,chemotherapeutic agents, such as anti-metabolic agents, e.g., Ara AC,5-FU and methotrexate, antimitotic agents, e.g. TAXOL, inblastine andvincristine, alkylating agents, e.g., melphalan, BCNU and nitrogenmustard, topoisomerase II inhibitors, e.g., VW-26, topotecan andBleomycin, strand-breaking agents, e.g., doxorubicin and DHAD,cross-linking agents, e.g., cisplatin and CBDCA, radiation andultraviolet light.

As used herein, the term “chemotherapeutic agent” is intended to includechemical reagents which inhibit the growth of proliferating cells ortissues wherein the growth of such cells or tissues is undesirable.Particular chemotherapeutic agents include, but are not limited to (i)antimetabolites, such as cytarabine, fludarabine,5-fluoro-2′-deoxyuridine, gemcitabine, hydroxyurea or methotrexate; (ii)DNA-fragmenting agents, such as bleomycin, (iii) DNA-crosslinkingagents, such as chlorambucil, cisplatin, cyclophosphamide or nitrogenmustard; (iv) intercalating agents such as adriamycin (doxorubicin) ormitoxantrone; (v) protein synthesis inhibitors, such as L-asparaginase,cycloheximide, puromycin or diphtheria toxin; (Vi) topoisomerase 1poisons, such as camptothecin or topotecan; (vii) topoisomerase IIpoisons, such as etoposide (VP-16) or teniposide; (viii)microtubule-directed agents, such as colcemid, colchicine, paclitaxel,vinblastine or vincristine; (ix) kinase inhibitors such as flavopiridol,staurosporin, STI571 (CPG 57148B) or UCN-01 (7-hydroxystaurosporine);(x) enhancers of the AMPK signaling pathway, (xi) inhibitors of thePI3K/AKT/mTORC1 signaling pathway, (xii) inhibitors of the MEK/ERKsignaling pathway, (xiii) miscellaneous investigational agents such asthioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl transferaseinhibitors (L-739749, L-744832); polyphenols such as quercetin,resveratrol, piceatannol, epigallocatechine gallate, theaflavins,flavanols, procyanidins, betulinic acid and derivatives thereof; (xiv)hormones such as glucocorticoids or fenretinide; and (xv) hormoneantagonists, such as tamoxifen, finasteride or LHRH antagonists. In anembodiment, the chemotherapeutic compound is one or more of gemcitabine,cisplatin, doxorubicin, daunarubicin, paclitaxel, taxotere and mitomycinC. In a particular embodiment, the chemotherapeutic compound is one ormore of gemcitabine, cisplatin and paclitaxel.

Chemotherapeutic agents are well known in the art (see e.g., Gilman A.G., et al., The Pharmacological Basis of Therapeutics, 8th Ed., Sec12:1202-1263 (1990)), and are typically used to treat neoplasticdiseases. The chemotherapeutic agents generally employed in chemotherapytreatments are listed below in Table 1.

TABLE 1 NONPROPRIETARY NAMES CLASS TYPE OF AGENT (OTHER NAMES)Alkylating Nitrogen Mustards Mechlorethamine (HN₂) CyclophosphamideIfosfamide Melphalan (L-sarcolysin) Chlorambucil EthyleniminesHexamethylmelamine And Methylmelamines Thiotepa Alkyl SulfonatesBusulfan Alkylating Nitrosoureas Carmustine (BCNU) Lomustine (CCNU)Semustine (methyl-CCNU) Streptozocin (streptozotocin) TriazenesDecarbazine (DTIC; imethyltriazenoimidazolecarboxamide) Alkylatorcis-diamminedichloroplatinum II (CDDP) Antimetabolites Folic AcidAnalogs Methotrexate (amethopterin) Pyrimidine Analogs Fluorouracil(′5-fluorouracil; 5-FU) Floxuridine (fluorode-oxyuridine; FUdR)Cytarabine (cytosine arabinoside) gemcitabine (deoxycytidine analog)Purine Analogs Mercaptopuine (6-mercaptopurine; 6-MP) and RelatedThioguanine (6-thioguanine; TG) Inhibitors Pentostatin(2′-deoxycoformycin) Vinca Alkaloids Vinblastin (VLB) VincristineTopoisomerase Inhibitors Etoposide Teniposide Camptothecin Topotecan9-amino-campotothecin CPT-11 Natural Antibiotics Dactinomycin(actinomycin D) Products Adriamycin (Doxorubicin) Daunorubicin(daunomycin; rubindomycin) Doxorubicin Bleomycin Plicamycin(mithramycin) Mitomycin (mitomycin C) TAXOL (paclitaxel) TaxotereEnzymes L-Asparaginase Biological Response Interfon alfa Modifiersinterleukin 2 Misc. Agents Platinum Coordinationcis-diamminedichloroplatinum Complexes II (CDDP) Carboplatin OxaliplatinCisplatin Anthracendione Mitoxantrone Substituted Urea HydroxyureaMethyl Hydraxzine Procarbazine (N-methylhydrazine, Derivative (MIH)Adrenocortical Mitotane (o,p′-DDD) Suppressant AminoglutethimideHormones and Adrenocorticosteroids Prednisone Antagonists DexamethasoneProgestins Hydroxyprogesterone Caproate Medroxyprogesterone AcetateMegestrol acetate Estrogens Diethylstilbestrol Ethinyl estradiolAntiestrogen Tamoxifen Androgens Testosterone propionate FluoxymesteroneAntiandrogen Flutamide Gonadotropin-releasing Leuprolide Hormone analog

The chemotherapeutic agents used in the present methods can be a singleagent or a combination of agents. Preferred combinations will includeagents that have different mechanisms of action, e.g., the use of ananti-mitotic agent in combination with an alkylating agent.

As used herein, the term “pro-survival” or “pro-growth” pathways referto signaling pathways used by cancer cells to promote their growthand/or survival. The term “pathway” is intended to mean a set of systemcomponents involved in two or more sequential molecular interactionsthat result in the production of a product or activity. A pathway canproduce a variety of products or activities that can include, forexample, intermolecular interactions, changes in expression of a nucleicacid or polypeptide, the formation or dissociation of complex betweentwo or more molecules, accumulation or destruction of a metabolicproduct, activation or deactivation of an enzyme or binding activity.Thus, the term “pathway” includes a variety of pathway types, such as,for example, a biochemical pathway, a gene expression pathway, and aregulatory pathway. Similarly, a pathway can include a combination ofthese exemplary pathway types. Intracellular signaling via severalpathways, such as AMPK, PI3K/AKT, MEK/ERK, and JAK/STAT signalingpathways, leading to the activation of anti-apoptotic proteins and theinactivation of pro-apoptotic proteins (reviewed in Henson and Gibson,2006, Cellular Signaling 18:2089-2097). Without being bound by theory,it is believed that ceramide increases apoptosis in cancer cells andsuch desired cancer cell death is antagonized by pro-survival pathways.Accordingly, therapeutic agents that inhibit such pro-survival pathwaysare contemplated as useful therapeutic agents in combination withceramide.

In one embodiment, the pro-survival pathway is the AMP-activated proteinkinase (AMPK) signaling pathway. AMPK has a role in regulating the mTORpathway. Mammalian target of rapamycin (mTOR) is a serine/threoninekinase and a key regulator of protein synthesis. To inhibit cell growthand protect cells from apoptosis induced by glucose starvation, AMPKphosphorylates TSC2 at Thr-1227 and Ser-1345 increasing the activity ofthe TSC1 and TSC-2 complex to inhibit m-TOR. In addition, AMPK inhibitsmTOR action by phosphorylation on Thr-2446. Thus, AMPK indirectly anddirectly inhibits the activity of mTOR to limit protein synthesis. AMPKmay also be a therapeutic target for many cancers that have constitutiveactivation of the PI3K-Akt signaling pathway. Treatment of variouscancer cell lines by AICAR attenuated the cell proliferation bothin-vitro and in-vivo studies (Rattan et al., JBC 280, 39582 (2005)).Reports link the treatment of metformin with a lower risk of cancer indiabetic patients (Evans et al., BMJ 330, 1304 (2005)). The activationof AMPK by AICAR has been shown to reduce expression of the lipogenicenzymes FAS and ACC, resulting in suppression of proliferation inprostate cancer cells. Many cancer cells display a markedly increasedrate of de novo fatty acid synthesis correlated with high levels of FAS.Inhibition of FAS suppresses cancer cell proliferation and induces celldeath. Thus, AMPK activation and inhibition of FAS activity is a cleartarget for pharmacological therapy of cancers.

In another embodiment, the pro-survival pathway is the PI3K/AKT/mTORC1signaling pathway. The “PI3K/AKT signaling pathway” or “AKT signalingpathway” refers to one of the intracellular signaling pathways activatedby the binding of growth factors to receptor tyrosine kinases. Onactivation, PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate(PIP2) to phosphatidylinositol-3,4,5-triphosphate (PIP3), a process thatis reversed by PTEN. PIP3 signals activate the kinase PDK1, which inturn activates the kinase AKT. The AKT protein family, which members arealso called protein kinases B (PKB) plays an important role in mammaliancellular signaling. Akt kinase is a serine/threonine kinase which is adownstream effector molecule of phosphoinositide 3-kinase and isinvolved in protecting a cell from apoptosis. Akt kinase is thought tobe involved in the progression of cancer because it stimulates cellproliferation and suppresses apoptosis. Akt1 is involved in cellularsurvival pathways, by inhibiting apoptotic processes. Akt1 is also ableto induce protein synthesis pathways, and is therefore a key signalingprotein in the cellular pathways that lead to skeletal musclehypertrophy, and general tissue growth. Since it can block apoptosis,and thereby promote cell survival, Akt1 has been implicated as a majorfactor in many types of cancer. Akt is known to play a role in the cellcycle. Under various circumstances, activation of Akt was shown toovercome cell cycle arrest in G1 and G2 phases. Moreover, activated Aktmay enable proliferation and survival of cells that have sustained apotentially mutagenic impact and, therefore, may contribute toacquisition of mutations in other genes. AKT (activation, amplification)and PTEN (mutation, deletion, epigenetic inactivation) are deregulatedin many human cancers (Altomare et al., 2003, J. Cell Biochem.88:470-476; Ruggeri et al., 1998, Mol. Carcinog. 21:81-86; Cheng et al.,1996, Proc. Natl. Acad. Sci. USA 93:3636-3641; Staal et al., 1987, Proc.Natl. Acad. Sci. USA 84:5034-5037; Li et al., 2005, World J.Gastroenterol. 11:285-288; Li et al., 1997, Science 275:1943-1947; Goelet al., 2004, 64:3014-3012), PI3K pathway activation can be assessed byimmunohistochemical analysis of PTEN or phosphorylated AKT levels inclinical samples (Slipicevic et al., 2005, Am. J. Clin. Pathol.124:528-536). Molecular targets of such inhibitors include, but are notlimited to, PI3K, AKT, mTORC1, mTORC2, PDK1, MYC, cMET, FGFR2, growthfactors (EGF, b-FGF, IGF1, Insulin, or Heregulin) and the like. Othermolecular targets are well known in the art and are described, forexample in US 2011-0015869.

Exemplary inhibitors of PI3K/AKT signaling are also well known in theart and include, but are not limited to: phosphatidylinositol etherlipid analogs, alkylphospholipid analogs, allosteric AKT inhibitors,HSP90 inhibitor, alkylphospholipid perifosine, rapamycin, RAD001,FTY720, PDK1 inhibitors (BX-795, BX-912, and BX-320 (Feldman et al.,2005, J. Biol. Chem. 280:19867-19874); 7-hydroxystaurosporine (Sato etal., 2002, Oncogene, 21:1727-1738)); PI3K inhibitors (wortmannin (Wymannet al., 1996, Mol. Cell. Biol. 16:1722-1733); LY294002 (Vlahos et al.,1994, J. Biol. Chem. 269:5241-5248; Wetzker and Rommel, 2004, Curr.Pharm. Des. 10:1915-1922); IC87114 (Finan and Thomas, 2004, Biochem.Soc. Trans. 32:378-382; WO0181346); WO01372557; U.S. Pat. No. 6,403,588;WO0143266); AKT antibodies (Shin et al., 2005, Cancer Res. 65:2815-2824)(see also Cheng et al., Oncogene, 2005, 24:7482-7492 for review oninhibitors of AKT pathway), and IGFIR inhibitors (such as monoclonalantibody MK-0646 U.S. Pat. No. 7,241,444). The inhibitors and agentslisted in the Examples section that were used to identify and refine thegrowth factor signaling pathway biomarkers are also exemplary growthfactor pathway agents (i.e., AKT1/2 inhibitors L-001154547 ('547;3-phenyl-2-(4-{[4-(5-pyridin-2-yl-1H-1,2,4-triazol-3-yl)piperidin-1-yl]me-thyl}phenyl)-1,6-naphthyridin-5(6H)-one;disclosed in WO2006065601), L-01173931 ('931;6-Methyl-3-phenyl-2-(4-{[4-(5-pyridin-2-yl-1H-1,2,4-triazol-3-yl)piperidi-n-1-yl]-methyl}phenyl)-1,6-naphthyridin-5(6H)-one;disclosed in WO2006065601; gamma secretase inhibitor 421B (U.S. Pat. No.7,138,400 and WO02/36555); cMET inhibitors L-001501404(4-(6-Phenyl-[1,2,4]triazolo[4,3-b][1,2,4]triazin-3-ylmethyl)-phenol,see also U.S. Pat. No. 7,122,548), MK-2461(N-[(2R)-1,4-dioxan-2-ylmethyl]-N-methyl-N′-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide),and L-001793225(1-[3-(1-Methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[1,2-b]pyri-din-7-yl]-N-(pyridin-2-ylmethyl)methanesulfonamide.

In still another embodiment, the pro-survival pathway is the MEK/ERKsignaling pathway. The extracellular signal regulated kinases (ERKs) areactivated by multiple signals including growth factors, cytokines,transforming growth factors, and G protein-coupled receptors. Thesesignals lead to activation of RAS small G proteins which activate RAFkinases. Active RAF kinases phosphorylate and activate MEK kinases,which subsequently phosphorylate and activate ERK1/2 kinases. ERK1/2kinases phosphorylate and regulate numerous substrates including otherprotein kinases, protein phosphatases, transcription factors,scaffolding proteins, signaling molecules and apoptosis-related proteinswhich lead to a variety of cell type and context-dependent responses.Constitutive activation of ERK1/2 by activating mutations in NRAS orBRAF is observed in the majority of melanomas and plays an integral rolein the regulation of proliferation, invasiveness, and survival. In oneembodiment, “ERK signaling” is signaling involving or mediated by thekinase activity of ERK1/2 kinases. In another embodiment, ERK signalingcomprises signal transduction via downstream targets of ERK1/2 kinaseactivity. Components of the ERK signaling pathway are known to those ofordinary skill in the art. For example, in humans, components of the ERKsignaling pathway that can positively regulate ERK signaling include,for example, BRAF (NCBI Gene ID No: 673); EGFR (NCBI Gene ID No: 1956);HER2 (NCBI Gene ID No: 2064); c-KIT (NCBI Gene ID No: 3815); MET (NCBIGene ID No: 4233); MEK1 (NCBI Gene ID No: 5604); MEK2 (NCBI Gene ID No:5605); ERK1 (NCBI Gene ID No: 5595); ERK2 (NCBI Gene ID No: 5594); HRAS(NCBI Gene ID No: 3265); KRAS (NCBI Gene ID No: 3845); and NRAS (NCBIGene ID No: 4893).

An inhibitor of ERK signaling can be an antagonist of any component ofthe ERK signaling pathway that positively regulates ERK signaling, e.g.BRAF or MEK, or an agent which decreases the amount or activity of thosecomponents, e.g., an RNAi molecule. An inhibitor of ERK signaling can bean agonist of any component of the ERK signaling pathway whichnegatively regulates ERK signaling, or an agent which increases theamount or activity of those components. In some embodiments, aninhibitor of ERK signaling specifically inhibits the kinase activity ofone or more RAF kinases or an ortholog thereof, e.g., it decreases thephosphorylation of one or more MEK kinases. In some embodiments, aninhibitor of ERK signaling is a specific inhibitor of the activity ofBRAF. In some embodiments, an inhibitor of ERK signaling is a specificinhibitor of the activity of a mutant form of BRAF. In some embodiments,an inhibitor of ERK signaling is a specific inhibitor of the activity ofBRAF.sup. V600E. In some embodiments, an inhibitor of ERK signalingspecifically inhibits the kinase activity of one or more MEK kinase oran ortholog thereof, e.g., it decreases the phosphorylation of ERK1/2.In some embodiments, an inhibitor of ERK signaling specifically inhibitsthe kinase activity of one or more of ERK1 and ERK2 kinases or anortholog thereof, e.g., it decreases the phosphorylation of a substrateof ERK1/2. Inhibition of ERK signaling can be measured according tomethods well-known to those of ordinary skill in the art. By way ofnon-limiting example, inhibition of ERK signaling can be measured bydetermining the level of dual-phosphorylated ERK1/2 (ppERK1/2) asdescribed in detail elsewhere herein. In brief, the level of ppERK1/2can be detected by immunoblot assay. Contacting a cell with an agentthat is an inhibitor of ERK signaling will cause the cell to exhibit alower level of ppERK1/2 than a cell not contacted with the agent.Components of the ERK signaling pathway that can negatively regulate ERKsignaling include, for example, SGK1 (NCBI Gene ID No: 6446); IGFBP7(NCBI Gene ID No: 3490); SPRED1 (NCBI Gene ID No: 161742); and KSR1(NCBI Gene ID No: 8844).

In some embodiments, the inhibitor of ERK signaling can be an inhibitorof MEK. As used herein, the term “inhibitor of MEK” refers to a compoundor agent, such as a small molecule, that inhibits, decreases, lowers, orreduces the activity of MEK. Examples of inhibitors of MEK include, butare not limited to, AZD6244(6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimida-zole-5-carboxylicacid (2-hydroxy-ethoxy)-amide; selumetinib; Structure IV), and U0126(1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio]butadiene;ARRY-142886; Structure V). Further non-limiting examples of MEKinhibitors include PD0325901, AZD2171, GDC-0973/XL-518, PD98059,PD184352, GSK1120212, RDEA436, RDEA119/BAY869766, AS703026, BIX 02188,BIX 02189, CI-1040 (PD184352), PD0325901, and PD98059. These and otherinhibitors of MEK, as well as non-limiting examples of their methods ofmanufacture, are described in U.S. Pat. Nos. 5,525,625; 6,251,943;7,820,664; 6,809,106; 7,759,518; 7,485,643; 7,576,072; 7,923,456;7,732,616, 7,271,178; 7,429,667; 6,649,640; 6,495,582; 7,001,905; USPatent Publication No. US2010/0331334, US2009/0143389, US2008/0280957,US2007/0049591, US2011/0118298, International Patent ApplicationPublication No. WO98/43960, WO99/01421, WO99/01426, WO00/41505,WO00/42002, WO00/42003, WO00/41994, WO00/42022, WO00/42029, WO00/68201,WO01/68619, WO02/06213 and WO03/077914, the contents of which are hereinincorporated by reference in their entireties.

In another embodiment, a therapeutic agent is an inhibitor of EGFR.Epidermal Growth Factor Receptor (EGFR) is a member of the type 1subgroup of receptor tyrosine kinase family of growth factor receptorswhich play critical roles in cellular growth, differentiation andsurvival. Activation of these receptors typically occurs via specificligand binding which results in hetero- or homodimerization betweenreceptor family members, which subsequent autophosphorylation of thetyrosine kinase domain. Specific ligands which bind to EGFR includeepidermal growth factor (EGF), transforming growth factor alpha (TGFalpha), amphiregulin and some viral growth factors. Activation of EGFRtriggers a cascade of intracellular signaling pathways involved in bothcellular proliferation (the ras/raf/MAP kinase pathway) and survival(the PI3 kinase/Akt pathway). Members of this family, including EGFR andHER2, have been directly implicated in cellular transformation. A numberof human malignancies are associated with aberrant or overexpression ofEGFR and/or overexpression of its specific ligands. Gullick, Br. Med.Bull. (1991), 74:87-98; Modijtahedi & Dean, Int. J. Oncol. (1994),4:277-96; Salomon, et al., Crit. Rev. Oncol. Hematol. (1995),19:183-232. Aberrant or overexpression of EGFR has been associated withan adverse prognosis in a number of human cancers, including head andneck, breast, colon, prostate, lung (e.g., NSCLC, adenocarcinoma andsquamous lung cancer), ovarian, gastrointestinal cancers (gastric,colon, pancreatic), renal cell cancer, bladder cancer, glioma,gynecological carcinomas and prostate cancer. In some instances,overexpression of tumor EGFR has been correlated with bothchemoresistance and a poor prognosis. Lei, et al., Anti-cancer Res.(1999), 19:221-28; Veale, et al., Br. J. Cancer (1993); 68:162-65.Mutations in EGFR are associated with many types of cancer as well. Forexample, EGFR mutations are highly prevalent in non-mucinous BACpatients. Finberg, et al., J. Mol. Diagnostics. (2007) 9(3):320-26. Inan embodiment the EGFR inhibitor is an antibody such as Erbitutux™(cetuximab, Imelone Systems Inc.) and ABX-EGF (panitumumab, Abgenix,Inc.). In another embodiment the EGFR inhibitor is a small molecule thatcompetes with ATP such as Tarceva™ (erlotinib, OSI Pharmaceuticals),Iressa™ (gefitinib, Astra-Zeneca), tyrphostins described by Dvir, etal., J Cell Biol., 113:857-865 (1991); tricyclic pyrimidine compoundsdisclosed in U.S. Pat. No. 5,679,683; compound6-(2,6-dichlorophenyl)-2-(4-(2-diethylaminoethoxy)phenylamino)-8-methyl-8H-pyrido(2,3-d)pyrimidin-7-one(known as PD166285) disclosed in Panek, et al., Journal of Pharmacologyand Experimental Therapeutics 283, 1433-1444 (1997).

In addition to the specific agents described above, it is furthercontemplated that a polypeptide, an antibody or antigen binding fragmentthereof, a toxin, and RNA interfering molecule, an siRNA molecule, andshRNA molecule, an antisense oligonucleotide, a peptide, apeptidomimetic, an aptamer, and the like, as well as combinationsthereof, that appropriately enhance or inhibit the targets ofpro-survival signaling pathways can also be used as a therapeutic agentaccording to the present invention. In particular, the nucleic acidsequence, amino acid sequence, functional domain, structural domain,gene locus, and other identifying information for the signaling pathwaytargets described herein are well known in the art. For example, KRASnucleic acid and amino acid sequences from many organisms is well knownin the art and include, for example, canine KRAS (NCBI AccessionXM_540523.3, XP_540523.3, XM_003432429.1, and XP_00343247.1), chimpanzeeKRAS (NCBI Accession XM_003313794.1, XP_003313842.1, XM_528758.3, andXP_528758.3), cow KRAS (NCBI Accession NM_001110001.1 andNP_001103471.1), mouse KRAS (NCBI Accession NM_021284.6 andNP_067259.4), rat KRAS (NCBI Accession NM_031515.3 and NP_113703.1),chicken KRAS (NCBI Accession NM_001256162.1 and NP_001243091.1), andzebrafish KRAS (NCBI Accession NM_001003744.1 and NP_001003744.1). HumanKRAS sequences include the following:

KRAS isoform a coding nucleic acid sequence (NCBI Accession NM_033360.2)SEQ ID NO 1:

  1 atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag tgccttgacg 61 atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga ggattcctac121 aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagcaggt181 caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg ctttctttgt241 gtatttgcca taaataatac taaatcattt gaagatattc accattatag agaacaaatt301 aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa atgtgatttg361 ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta tggaattcct421 tttattgaaa catcagcaaa gacaagacag agagtggagg atgcttttta tggaattcct481 agggagatcc gacaatacag attgaaaaaa atcagcaaag aagaaaagac tcctggctgt541 gtgaaaatta aaaaatgcat tataatgtaa

KRAS isoform an amino acid sequence (NCBI Accession NM_203524.1) SEQ IDNO 2:

  1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkqvvidget clldildtag 61 geeysamrdq ymrtgegflc fvainntkef edihhyreqi krvkdsedvp mvlvgnkccl121 psrtvdtkga qdlareygip fietsaktrq rvedafytlv reirqyrlkk iskeektpgc181 vkikkciim

KRAS isoform b coding nucleic acid sequence (NCBI Accession NM_004985.3)SEQ ID NO 3:

  1 atgactgaat ataaacttgt ggtagttgga gctggtggcg taggcaagag tgccttgacg 61 atacagctaa ttcagaatca ttttgtggac gaatatgatc caacaataga ggattcctac121 aggaagcaag tagtaattga tggagaaacc tgtctcttgg atattctcga cacagcaggt181 caagaggagt acagtgcaat gagggaccag tacatgagga ctggggaggg ctttctttgc241 gtatttgcca taaataatac taaatcattt gaagatattc accattatag agaacaaatt301 aaaagagtta aggactctga agatgtacct atggtcctag taggaaataa atgtgatttg361 ccttctagaa cagtagacac aaaacaggct caggacttag caagaagtta tggaattcct421 tttattgaaa catcagcaaa gacaagacag ggtgttgatg atgccttcta tacattagtt481 cgagaaattc gaaaacataa agaaaagatg agcaaagatg gtaaaaagaa gaaaaagaag541 tcaaagacaa agtgtgtaat tatgtaa

KRAS isoform b amino acid sequence (NCBI Accession NP_004976.2) SEQ IDNO 4:

  1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkgvvidget clldildtag 61 qeeysamrdq ymrtgegflc vfainntksf edihhyreqi krvkdsedvp mvlvgnkcdl121 psrtvdtkqa qdlareygip fietsaktrq gvddafytlv reirkhkekm skdgkkkkk 181sktkcyim

The present invention further provides variants, fragments, andfunctionally similar homologs of the pro-survival signaling pathwaytargets for enhanced or inhibited activity according to the methodsdescribed further herein. For example, a nucleic acid molecule of such atarget can comprise only a portion of a nucleic acid sequence needed toalter target activity. For example, such nucleic acid molecules canencode a polypeptide that contains changes in amino acid residues thatare not essential for activity. Such polypeptides differ in amino acidsequence from the naturally-occurring proteins, yet retain biologicalactivity. In one embodiment, such a protein has an amino acid sequencethat is at least about 40% identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%,87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical tothe amino acid sequence of one of the target proteins. An isolatednucleic acid molecule encoding a variant protein can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of nucleic acids of the invention, suchthat one or more amino acid residue substitutions, additions, ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art.

In some embodiments, antisense nucleic acid molecules, i.e., moleculeswhich are complementary to a sense nucleic acid target, e.g.,complementary to the coding strand of a double-stranded cDNA or mRNAmolecule, is useful. Accordingly, an antisense nucleic acid molecule canhydrogen bond to (i.e. anneal with) a sense nucleic acid target. Theantisense nucleic acid can be complementary to an entire coding strand,or to only a portion thereof, e.g., all or part of the protein codingregion (or open reading frame). An antisense nucleic acid molecule canalso be antisense to all or part of a non-coding region of the codingstrand of a nucleotide sequence encoding a polypeptide of the invention.The non-coding regions (“5′ and 3′ untranslated regions”) are the 5′ and3′ sequences which flank the coding region and are not translated intoamino acids. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. Anantisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense nucleic acid molecules of the inventionare typically administered to a subject or generated in situ such thatthey hybridize with or bind to cellular mRNA and/or genomic DNA encodinga polypeptide corresponding to a selected marker of the invention tothereby inhibit expression of the marker, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. Examples of a route of administration of antisensenucleic acid molecules of the invention includes direct injection at atissue site or infusion of the antisense nucleic acid into a blood- orbone marrow-associated body fluid. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For example, for systemic administration, antisensemolecules can be modified such that they specifically bind to receptorsor antigens expressed on a selected cell surface, e.g., by linking theantisense nucleic acid molecules to peptides or antibodies which bind tocell surface receptors or antigens. The antisense nucleic acid moleculescan also be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

An antisense nucleic acid molecule of the invention can be an α-anomericnucleic acid molecule. An α-anomeric nucleic acid molecule formsspecific double-stranded hybrids with complementary RNA in which,contrary to the usual α-units, the strands run parallel to each other(Gaultier et al., 1987, Nucleic Acids Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-α-methylribonucleotide(Inoue et al., 1987, Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).

An “RNA interfering agent” as used herein, is defined as any agent whichinterferes with or inhibits expression of a target gene, e.g., a markerof the invention, by RNA interference (RNAi). Such RNA interferingagents include, but are not limited to, nucleic acid molecules includingRNA molecules which are homologous to the target gene, e.g., a marker ofthe invention, or a fragment thereof, short interfering RNA (siRNA), andsmall molecules which interfere with or inhibit expression of a targetgene by RNA interference (RNAi).

“RNA interference (RNAi)” is an evolutionally conserved process wherebythe expression or introduction of RNA of a sequence that is identical orhighly similar to a target gene results in the sequence specificdegradation or specific post-transcriptional gene silencing (PTGS) ofmessenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G.and Cullen, B. (2002) J. of Virology 76(18):9225), thereby inhibitingexpression of the target gene (see, e.g., U.S. Patent Application Nos:20030153519A1; 20030167490A1; and U.S. Pat. Nos. 6,506,559; 6,573,099,which are herein incorporated by reference in their entirety). In oneembodiment, the RNA is double stranded RNA (dsRNA). This process hasbeen described in plants, invertebrates, and mammalian cells. In nature,RNAi is initiated by the dsRNA-specific endonuclease Dicer, whichpromotes processive cleavage of long dsRNA into double-strandedfragments termed siRNAs. siRNAs are incorporated into a protein complexthat recognizes and cleaves target mRNAs. RNAi can also be initiated byintroducing nucleic acid molecules, e.g., synthetic siRNAs or RNAinterfering agents, to inhibit or silence the expression of targetgenes. As used herein, “inhibition of target gene expression” or“inhibition of marker gene expression” includes any decrease inexpression or protein activity or level of the target gene (e.g., amarker gene of the invention) or protein encoded by the target gene,e.g., a marker protein of the invention. The decrease may be of at least30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to theexpression of a target gene or the activity or level of the proteinencoded by a target gene which has not been targeted by an RNAinterfering agent.

The present invention also contemplates “short interfering RNA” (siRNA),also referred to herein as “small interfering RNA.” Such a molecule isdefined as an agent which functions to inhibit expression of a targetgene, e.g., by RNAi. As used herein, the term siRNA is intended to beequivalent to any term in the art defined as a molecule capable ofmediating sequence-specific RNAi. Such equivalents include, for example,double-stranded RNA (dsRNA), microRNA (mRNA), short hairpin RNA (shRNA),short interfering oligonucleotide, and post-transcriptional genesilencing RNA (ptgsRNA). An siRNA may be chemically synthesized, may beproduced by in vitro transcription, or may be produced within a hostcell. In one embodiment, siRNA is a double stranded RNA (dsRNA) moleculeof about 15 to about 40 nucleotides in length, preferably about 15 toabout 28 nucleotides, more preferably about 19 to about 25 nucleotidesin length, and more preferably about 19, 20, 21, or 22 nucleotides inlength, and may contain a 3′ and/or 5′ overhang on each strand having alength of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of theoverhang is independent between the two strands, i.e., the length of theoverhang on one strand is not dependent on the length of the overhang onthe second strand. Preferably the siRNA is capable of promoting RNAinterference through degradation or specific post-transcriptional genesilencing (PTGS) of the target messenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stemloop) RNA (shRNA). In one embodiment, these shRNAs are composed of ashort (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9nucleotide loop, and the analogous sense strand. Alternatively, thesense strand may precede the nucleotide loop structure and the antisensestrand may follow. These shRNAs may be contained in plasmids,retroviruses, and lentiviruses and expressed from, for example, the polIII U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003)RNA April; 9(4):493-501 incorporated by reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to asubject having or at risk for having cancer, to inhibit expression of amarker gene of the invention, e.g., a marker gene which is overexpressedin cancer (such as the markers listed in Table 3) and thereby treat,prevent, or inhibit cancer in the subject.

The present invention also encompasses ribozymes. Ribozymes arecatalytic RNA molecules with ribonuclease activity which are capable ofcleaving a single-stranded nucleic acid, such as an mRNA, to which theyhave a complementary region. Thus, ribozymes (e.g., hammerhead ribozymesas described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can beused to catalytically cleave mRNA transcripts to thereby inhibittranslation of the protein encoded by the mRNA. A ribozyme havingspecificity for a nucleic acid molecule encoding a target polypeptidecan be designed based upon the nucleotide sequence of a cDNAcorresponding to the marker. For example, a derivative of a TetrahymenaL-19 IVS RNA can be constructed in which the nucleotide sequence of theactive site is complementary to the nucleotide sequence to be cleaved(see Cech et al. U.S. Pat. No. 4,987,071; Cech et al. U.S. Pat. No.5,116,742). Alternatively, an mRNA encoding a polypeptide of theinvention can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules (see, e.g., Barteland Szostak, 1993, Science 261:1411-1418).

The present invention also encompasses nucleic acid molecules which formtriple helical structures. For example, expression of a polypeptide ofthe invention can be inhibited by targeting nucleotide sequencescomplementary to the regulatory region of the gene encoding thepolypeptide (e.g., the promoter and/or enhancer) to form triple helicalstructures that prevent transcription of the gene in target cells. Seegenerally Helene (1991) Anticancer Drug. Des. 6(6):569-84; Helene (1992)Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays14(12):807-15.

In various embodiments, nucleic acid molecules can be modified at thebase moiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acid molecules (see Hyrup et al.,1996, Bioorganic & Medicinal Chemistry 4(1): 5-23). As used herein, theterms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics,e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. Forexample, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, e.g., inducingtranscription or translation arrest or inhibiting replication. PNAs canalso be used, e.g., in the analysis of single base pair mutations in agene by, e.g., PNA directed PCR clamping; as artificial restrictionenzymes when used in combination with other enzymes, e.g., S1 nucleases(Hyrup (1996), supra; or as probes or primers for DNA sequence andhybridization (Hyrup, 1996, supra; Perry-O'Keefe et al., 1996, Proc.Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance theirstability or cellular uptake, by attaching lipophilic or other helpergroups to PNA, by the formation of PNA-DNA chimeras, or by the use ofliposomes or other techniques of drug delivery known in the art. Forexample, PNA-DNA chimeras can be generated which can combine theadvantageous properties of PNA and DNA. Such chimeras allow DNArecognition enzymes, e.g., RNASE H and DNA polymerases, to interact withthe DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup, 1996, supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63.For example, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs. Compounds such as 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite can be used as a link between the PNA and the 5′ end ofDNA (Mag et al., 1989, Nucleic Acids Res. 17:5973-88). PNA monomers arethen coupled in a step-wise manner to produce a chimeric molecule with a5′ PNA segment and a 3′ DNA segment (Finn et al., 1996, Nucleic AcidsRes. 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.,1975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (see, e.g., Krolet al., 1988, Bio/Techniques 6:958-976) or intercalating agents (see,e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The present invention also includes molecular beacon nucleic acidmolecules having at least one region which is complementary to a nucleicacid molecule of the invention, such that the molecular beacon us usefulfor quantitating the presence of the nucleic acid molecule of theinvention in a sample. A “molecular beacon” nucleic acid is a nucleicacid molecule comprising a pair of complementary regions and having afluorophore and a fluorescent quencher associated therewith. Thefluorophore and quencher are associated with different portions of thenucleic acid in such an orientation that when the complementary regionsare annealed with one another, fluorescence of the fluorophore isquenched by the quencher. When the complementary regions of the nucleicacid molecules are not annealed with one another, fluorescence of thefluorophore is quenched to a lesser degree. Molecular beacon nucleicacid molecules are described, for example, in U.S. Pat. No. 5,876,930.

In addition, aptamers are contemplated and can be produced using themethodology disclosed in a U.S. Pat. No. 5,270,163 and WO 91/19813.

The present invention also pertains to isolated proteins whichcorrespond to pro-survival signaling pathway targets and/orappropriately enhance or inhibit the activity of such targets andbiologically active portions thereof. In one embodiment, the nativepolypeptide corresponding to a biomarker can be isolated from cells ortissue sources by an appropriate purification scheme using standardprotein purification techniques. In another embodiment, polypeptidescorresponding to a biomarker of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, apolypeptide corresponding to a biomarker of the invention can besynthesized chemically using standard peptide synthesis techniques.Biologically active portions of a polypeptide corresponding to abiomarker of the invention include polypeptides comprising amino acidsequences sufficiently identical to or derived from the amino acidsequence of the protein corresponding to the biomarker which includefewer amino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding protein. A biologically active portionof a protein of the invention can be a polypeptide which is, forexample, 10, 25, 50, 100 or more amino acids in length. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated fro oneor more of the functional activities of the native form of a polypeptideof the invention.

Preferred polypeptides have an amino acid sequence of a protein encodedby a nucleic acid molecule encoding the target. Other useful proteinsare substantially identical (e.g., at least about 40%, preferably 50%,60%, 70%, 80%, 90%, 95%, or 99%) to one of these sequences and retainthe functional activity of the protein of the correspondingnaturally-occurring protein yet differ in amino acid sequence due tonatural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, word length=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, word length=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Suchan algorithm is incorporated into the ALIGN program (version 2.0) whichis part of the GCG sequence alignment software package. When utilizingthe ALIGN program for comparing amino acid sequences, a PAM120 weightresidue table, a gap length penalty of 12, and a gap penalty of 4 can beused. Yet another useful algorithm for identifying regions of localsequence similarity and alignment is the FASTA algorithm as described inPearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. Whenusing the FASTA algorithm for comparing nucleotide or amino acidsequences, a PAM120 weight residue table can, for example, be used witha k-tuple value of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

The invention also provides chimeric or fusion proteins. As used herein,a “chimeric protein” or “fusion protein” comprises all or part(preferably a biologically active part) of a polypeptide correspondingto a biomarker of the invention operably linked to a heterologouspolypeptide (i.e., a polypeptide other than the polypeptidecorresponding to the biomarker). Within the fusion protein, the term“operably linked” is intended to indicate that the polypeptide of theinvention and the heterologous polypeptide are fused in-frame to eachother. The heterologous polypeptide can be fused to the amino-terminusor the carboxyl-terminus of the polypeptide of the invention. Suchfusion proteins are well known in the art and include, for example,target proteins or polypeptides that enhance or inhibit the activity ofsuch target proteins fused to heterologous signal sequences, peptidetags, immunoglobulin fusion proteins, and the like. Chimeric and fusionproteins of the invention can be produced by standard recombinant DNAtechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see, e.g., Ausubel etal., supra). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST polypeptide).A nucleic acid encoding a polypeptide of the invention can be clonedinto such an expression vector such that the fusion moiety is linkedin-frame to the polypeptide of the invention.

The present invention also pertains to variants of the polypeptides.Such variants have an altered amino acid sequence which can function aseither agonists (mimetics) or as antagonists. Variants can be generatedby mutagenesis, e.g., discrete point mutation or truncation. An agonistcan retain substantially the same, or a subset, of the biologicalactivities of the naturally occurring form of the protein. An antagonistof a protein can inhibit one or more of the activities of the naturallyoccurring form of the protein by, for example, competitively binding toa downstream or upstream member of a cellular signaling cascade whichincludes the protein of interest. Thus, specific biological effects canbe elicited by treatment with a variant of limited function. Treatmentof a subject with a variant having a subset of the biological activitiesof the naturally occurring form of the protein can have fewer sideeffects in a subject relative to treatment with the naturally occurringform of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity. In oneembodiment, a variegated library of variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential protein sequences is expressible as individual polypeptides,or alternatively, as a set of larger fusion proteins (e.g., for phagedisplay). There are a variety of methods which can be used to producelibraries of potential variants of the polypeptides of the inventionfrom a degenerate oligonucleotide sequence. Methods for synthesizingdegenerate oligonucleotides are known in the art (see, e.g., Narang,1983, Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem.53:323; Itakura et al., 1984, Science 198:1056; Ike et al., 1983 NucleicAcid Res. 11:477).

In addition, libraries of fragments of the coding sequence of apolypeptide corresponding to a biomarker of the invention can be used togenerate a variegated population of polypeptides for screening andsubsequent selection of variants. For example, a library of codingsequence fragments can be generated by treating a double stranded PCRfragment of the coding sequence of interest with a nuclease underconditions wherein nicking occurs only about once per molecule,denaturing the double stranded DNA, renaturing the DNA to form doublestranded DNA which can include sense/antisense pairs from differentnicked products, removing single stranded portions from reformedduplexes by treatment with S1 nuclease, and ligating the resultingfragment library into an expression vector. By this method, anexpression library can be derived which encodes amino terminal andinternal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.The most widely used techniques, which are amenable to high throughputanalysis, for screening large gene libraries typically include cloningthe gene library into replicable expression vectors, transformingappropriate cells with the resulting library of vectors, and expressingthe combinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a techniquewhich enhances the frequency of functional mutants in the libraries, canbe used in combination with the screening assays to identify variants ofa protein of the invention (Arkin and Yourvan, 1992, Proc. Natl. Acad.Sci. USA 89:7811-7815; Delgrave et al., 1993, Protein Engineering6(3):327-331).

Also contemplated are antibodies that bind to pro-survival signalingpathway targets to thereby appropriately enhance or inhibit theiractivity. An isolated target polypeptide or a fragment thereof (or anucleic acid encoding such a polypeptide), can be used as an immunogento generate antibodies that bind to said immunogen, using standardtechniques for polyclonal and monoclonal antibody preparation.

Unless otherwise specified herein, the terms “antibody” and “antibodies”broadly encompass naturally-occurring forms of antibodies (e.g. IgG,IgA, IgM, IgE) and recombinant antibodies such as single-chainantibodies, chimeric and humanized antibodies and multi-specificantibodies, as well as fragments and derivatives of all of theforegoing, which fragments and derivatives have at least an antigenicbinding site. Antibody derivatives may comprise a protein or chemicalmoiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-bindingportion” of an antibody (or simply “antibody portion”). The term“antigen-binding portion”, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toa target antigen. It has been shown that the antigen-binding function ofan antibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentpolypeptides (known as single chain Fv (scFv); see e.g., Bird eta al.(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single chain antibodies are also intended to be encompassedwithin the term “antigen-binding portion” of an antibody. Any VH and VLsequences of specific scFv can be linked to human immunoglobulinconstant region cDNA or genomic sequences, in order to generateexpression vectors encoding complete IgG polypeptides or other isotypes.VH and VL can also be used in the generation of Fab, Fv or otherfragments of immunoglobulins using either protein chemistry orrecombinant DNA technology. Other forms of single chain antibodies, suchas diabodies are also encompassed. Diabodies are bivalent, bispecificantibodies in which VH and VL domains are expressed on a singlepolypeptide chain, but using a linker that is too short to allow forpairing between the two domains on the same chain, thereby forcing thedomains to pair with complementary domains of another chain and creatingtwo antigen binding sites (see e.g., Holliger, P., et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may bepart of larger immunoadhesion polypeptides, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionpolypeptides include use of the streptavidin core region to make atetrameric scFv polypeptide (Kipriyanov, S. M., et al. (1995) HumanAntibodies and Hybridomas 6:93-101) and use of a cysteine residue, amarker peptide and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv polypeptides (Kipriyanov, S. M., et al. (1994) Mol.Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventionaltechniques, such as papain or pepsin digestion, respectively, of wholeantibodies. Moreover, antibodies, antibody portions and immunoadhesionpolypeptides can be obtained using standard recombinant DNA techniques,as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, or syngeneic; ormodified forms thereof (e.g., humanized, chimeric, etc.). Antibodies mayalso be fully human. Preferably, antibodies of the invention bindspecifically or substantially specifically to pro-survival signalingpathway polypeptides of interest or fragments thereof. The terms“monoclonal antibodies” and “monoclonal antibody composition”, as usedherein, refer to a population of antibody polypeptides that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of an antigen, whereas the term “polyclonalantibodies” and “polyclonal antibody composition” refer to a populationof antibody polypeptides that contain multiple species of antigenbinding sites capable of interacting with a particular antigen. Amonoclonal antibody composition typically displays a single bindingaffinity for a particular antigen with which it immunoreacts.

C. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceuticallyacceptable compositions which comprise a therapeutically-effectiveamount of agents described herein, e.g., a chemotherapeutic agent,formulated together with one or more pharmaceutically acceptablecarriers (additives) and/or diluents. As described in detail below, thepharmaceutical compositions of the present invention may be speciallyformulated for administration in solid or liquid form, including thoseadapted for the following: (1) oral administration, for example,drenches (aqueous or non-aqueous solutions or suspensions), tablets,boluses, powders, granules, pastes; (2) parenteral administration, forexample, by subcutaneous, intramuscular or intravenous injection as, forexample, a sterile solution or suspension; (3) topical application, forexample, as a cream, ointment or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; or (5) aerosol, for example, as an aqueous aerosol, liposomalpreparation or solid particles containing the compound.

The term “administering” is intended to include routes of administrationwhich allow the compositions described herein to perform their intendedfunctions of treating cancer or inhibiting cancer cell growth. Examplesof routes of administration which can be used include injection(subcutaneous, intravenous, parenterally, intraperitoneally,intrathecal, etc.), oral, inhalation, and transdermal. The injection canbe bolus injections or can be continuous infusion. Depending on theroute of administration, the therapeutic agents can be coated with ordisposed in a selected material to protect it from natural conditionswhich may detrimentally effect its ability to perform its intendedfunction. The therapeutic agents can be administered alone, or inconjunction with a pharmaceutically acceptable carrier. Further thetherapeutic agents can be administered as a mixture of therapeuticagents, which also can be coadministered with a pharmaceuticallyacceptable carrier.

As used herein, an “effective amount,” when used with respect to thecombination of agents described herein includes, without limitation, anamount of each agent in the combination that provides a statisticallysignificant desired effect on cancer cells. In some embodiments, theeffect amount can be narrowed to further require clinical acceptabilityof the amount of toxicity to non-cancer cells. Representative desiredeffects are described herein. For example, the effect can be a decreasein the rate of tumor growth, a cessation of tumor growth, or a reductionin the size, mass, metabolic activity, or volume of the tumor, asmeasured by standard criteria such as, but not limited to, the ResponseEvaluation Criteria for Solid Tumors (RECIST), a statisticallysignificant increase in survival relative to treatment with individualagents of the combination or sub-combinations of the combination alone,and the like. The effective amount can vary depending on such factors asthe type of cell growth being treated or inhibited, the type oftherapeutic agent(s) employed, the particular therapeutic agent, thesize of the subject, or the severity of the cancer cell growth or tumor.For example, the choice of each of the individual agents which make upthe combination can affect what constitutes an “effective amount”. Oneof ordinary skill in the art would be able to study the aforementionedfactors and make the determination regarding the effective amount of thecombination of the therapeutic agents without undue experimentation.

For example, an in vitro assay can be used to determine an “effectiveamount” of the therapeutic agents. The ordinarily skilled artisan wouldselect an appropriate amount of each individual agent in the combinationfor use in the aforementioned in vitro assay. The cell survival fractioncan be used to determine whether the selected amounts were an “effectiveamount” for the particular combination of therapeutic agents. Forexample, the selected amounts used within the assay preferably shouldresult in a killing of at least 50% of the cells, more preferably 75%,and most preferably at least 95%. In a preferred embodiment, theeffective dose of the therapeutic agent is a subtoxic dose. As usedherein, the term subtoxic dose refers to a dose which results in thekilling of less than about 10% of the cells.

The regimen (e.g., order) of administration can also affect whatconstitutes an effective amount. Further, several divided dosages, aswell as staggered dosages, can be administered daily or sequentially, orthe dose can be continuously infused. Further, the dosages can beproportionally increased or decreased as indicated by the exigencies ofthe therapeutic situation.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose combinations of therapeutic agents, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “pharmaceutically-acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of the therapeuticagents encompassed by the invention. These salts can be prepared in situduring the final isolation and purification of the therapeutic agents,or by separately reacting a purified therapeutic agents in its free baseform with a suitable organic or inorganic acid, and isolating the saltthus formed. Representative salts include the hydrobromide,hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,valerate, oleate, palmitate, stearate, laurate, benzoate, lactate,phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate,napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonatesalts and the like. (See, for example, Berge et al. (1977)“Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19).

In other cases, the therapeutic agents useful in the methods of thepresent invention may contain one or more acidic functional groups and,thus, are capable of forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of therapeutic agents. These salts canlikewise be prepared in situ during the final isolation and purificationof the therapeutic agents, or by separately reacting the purifiedtherapeutic agents in its free acid form with a suitable base, such asthe hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptablemetal cation, with ammonia, or with a pharmaceutically-acceptableorganic primary, secondary or tertiary amine. Representative alkali oralkaline earth salts include the lithium, sodium, potassium, calcium,magnesium, and aluminum salts and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate; sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating, suchas citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association a therapeutic agent with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a therapeutic agent with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouth washes and the like, each containinga predetermined amount of a therapeutic agent as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically-acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (8) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-gilledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active therapeutic agents may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more therapeuticagents with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of atherapeutic agent include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically-acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition to atherapeutic agent, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to a therapeutic agent,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The therapeutic agent can be alternatively administered by aerosol. Thisis accomplished by preparing an aqueous aerosol, liposomal preparationor solid particles containing the compound. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a therapeutic agent to the body. Such dosage forms can bemade by dissolving or dispersing the agent in the proper medium.Absorption enhancers can also be used to increase the flux of thepeptidomimetic across the skin. The rate of such flux can be controlledby either providing a rate controlling membrane or dispersing thepeptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more therapeutic agents in combinationwith one or more pharmaceutically-acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben, chlorbutanol,phenol sorbic acid, and the like. It may also be desirable to includeisotonic agents, such as sugars, sodium chloride, and the like into thecompositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices of atherapeutic agent in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the therapeutic agents of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

III. Anti-Cancer Methods

The methods of the present invention relate to therapeutic andprophylactic compositions for treating cancer or preventing the growthof cancer cells, e.g., tumor growth in a subject. The compositions ofthe present invention include an effective amount of ceramide (e.g.,C6-ceramide) and an effective amount of one or more anti-cancer agents.Other aspects of the present invention include compositions, such aspackaged C6-ceramide and additional therapeutic agent(s). The packagedcompounds and agents can also include instructions for using thecomposition for treating cancer or preventing the growth of cancercells.

In another aspect, the invention relates to methods for treating cancer,e.g., inhibiting tumor growth, in a subject by administering to asubject an effective amount of a ceramide (e.g., C6-ceramide) and aneffective amount of a therapeutic agent, e.g., a chemotherapeutic agent,wherein the ceramide allows for a reduction in the amount of thetherapeutic agent(s) required to be effective, resulting in fewer sideeffects in the subject being treated.

In general, the methods of the present invention include a step ofcontacting cancer cells with a combination of a ceramide (e.g.,C6-ceramide) and a therapeutic agent, e.g., a chemotherapeutic agent,effective for promoting apoptosis or cell death. In some embodiments,the cancer cells can harbor activating KRAS mutations. In otherembodiments, the cancer cells do not harbor activating KRAS mutations.

As used herein, the term “cell death” includes the processes by whichmammalian cells die or become terminally differentiated. Such processesinclude apoptosis (both reversible and irreversible) and processesthought to involve apoptosis (e.g., cell senescence), as well asnecrosis and terminal cell differentiation. “Cell death” is used hereinto refer to the death or imminent death of nucleated cells (e.g.,neurons, myocytes, hepatocytes and the like) as well as to the death orimminent death of anucleate cells (e.g., red blood cells, platelets, andthe like). Cell death is typically manifested by the exposure of theinternal membrane phospholipid phosphatidylserine (PS) on the outerleaflet of the plasma membrane and can be detected by art recognizedmethods.

As used herein the term “apoptosis” includes programmed cell death whichcan also be detected using techniques which are known in the art. Forexample, apoptotic cell death can be characterized, e.g., by cellshrinkage, membrane blebbing and chromatin condensation culminating incell fragmentation. Cells undergoing apoptosis also display acharacteristic pattern of internucleosomal DNA cleavage. Apoptosis canbe measured in the presence or the absence of Fas-mediated signals. Inone embodiment, cytochrome C release from mitochondria during cellapoptosis can be detected, e.g., plasma cell apoptosis (as described in,for example, Bossy-Wetzel E. et al. (2000) Methods in Enzymol.322:235-42). Other assays include cytofluorometric quantitation ofnuclear apoptosis induced in a cell-free system (as described in, forexample, Lorenzo H. K. et al. (2000) Methods in Enzymol. 322:198-201);apoptotic nuclease assays (as described in, for example, Hughes F. M.(2000) Methods in Enzymol. 322:47-62); analysis of apoptotic cells, e.g.apoptotic plasma cells, by flow and laser scanning cytometry (asdescribed in, for example, Darzynkiewicz Z. et al. (2000) Methods inEnzymol. 322:18-39); detection of apoptosis by annexin V labeling (asdescribed in, for example, Bossy-Wetzel E. et al. (2000) Methods inEnzymol. 322:15-18); transient transfection assays for cell death genes(as described in, for example, Miura M. et al. (2000) Methods in Enzymol322:480-92); and assays that detect DNA cleavage in apoptotic cells,e.g., apoptotic plasma cells (as described in, for example, Kauffman S.H. et al. (2000) Methods in Enzymol. 322:3-15). Apoptosis can also bemeasured by propidium iodide staining or by TUNEL assay.

In another aspect, the invention features methods for inhibiting theproliferation of cancer cells by contacting the cells with a ceramide(e.g., C6-ceramide) and a therapeutic agent(s). In general, the methodincludes a step of contacting cancer cells with a ceramide (e.g.,C6-ceramide) and a therapeutic agent(s) effective for reducing theproliferation of cancer cells. The reduced proliferation of cancer cellscan be detected by at least one of the following biological activities:(1) a decrease in solid tumor cell proliferation; (2) a decrease in thefraction of cells in the DNA synthesis phase of the cell cycle(S-phase); (3) an increase in expression of differentiation-associatedmarkers; (4) a decrease in the expression of proliferation-associatedmarkers such as Ki-67 (MIB-1), e.g., a decrease in the expression ofKi-67 by about 30-50%, using techniques which are known in the art.Changes in expression can occur in the protein or mRNA levels.

The present method can be performed on cells in culture, e.g., ex vivo,or can be performed on cells, present in an animal subject, e.g., aspart of an in vivo therapeutic protocol. The therapeutic regimen can becarried out on a human or other animal subject.

The methods of the present invention allow for a reduction in the amountof the therapeutic agent, e.g., a chemotherapeutic agent, required to beeffective, resulting in fewer side effects in the subject being treated.

In one embodiment, the cells to be treated are pancreatic cancer and/orcolorectal cancer cells. For instance, the instant method can be carriedout to prevent the proliferation of a pancreatic cancer and/orcolorectal cancer cell tumor.

Determination of a therapeutically effective amount of a ceramide (e.g.,C6-ceramide) and a therapeutically effective amount of a therapeuticagent, e.g., a chemotherapeutic agent, can be readily made by thephysician (the “attending clinician”), as one skilled in the art, by theuse of known techniques and by observing results obtained underanalogous circumstances. The dosages may be varied depending upon therequirements of the patient in the judgment of the attending clinician,the severity of the condition being treated and the particular compoundbeing employed. In determining the therapeutically effective amount ordose, a number of factors are considered by the attending clinician,including, but not limited to: the specific hyperplastic/neoplastic cellinvolved; pharmacodynamic characteristics of the particular agent andits mode and route of administration; the desired time course oftreatment, the species of mammal; its size, age, and general health; thespecific disease involved; the degree of or involvement or the severityof the disease; the response of the individual patient; the particularcompound administered, the mode of administration; the bioavailabilitycharacteristics of the preparation administered; the dose regimenselected; the kind of concurrent treatment; and other relevantcircumstances. U.S. Pat. No. 5,427,916, for example, describes methodfor predicting the effectiveness of antineoplastic therapy in individualpatients, and illustrates certain methods which can be used inconjunction with the treatment protocols of the instant invention.

The effectiveness of any particular combination of a ceramide (e.g.,C6-ceramide) with a therapeutic agent(s) to treat cancer can bemonitored by comparing two or more samples obtained from a patientundergoing anti-cancer treatment. In general, it is preferable to obtaina first sample from the patient prior to beginning therapy and one ormore samples during treatment. In such a use, a baseline of expressionof cancer cells prior to therapy is determined and then changes in thebaseline state of expression of cancer cells is monitored during thecourse of therapy. Alternatively, two or more successive samplesobtained during treatment can be used without the need of apre-treatment baseline sample. In such a use, the first sample obtainedfrom the subject is used as a baseline for determining whether theexpression of cancer cells is increasing or decreasing.

In general, when monitoring the effectiveness of a therapeutictreatment, two or more samples from the patient are examined.Preferably, three or more successively obtained samples are used,including at least one pretreatment sample.

EXAMPLES Example 1: Materials and Methods Used in Examples 2-8

A. Chemicals and Reagents

C6-ceramide was provided by Avanti (Alabaster, AB, CN: 860506P).paclitaxel and gemcitabine were obtained from the pharmacy at RogerWilliams Medical Center. LY 294002. Rapamycin and U0126 were purchasedfrom CalbioChem (San Diego, Calif.). ERK1/2, AKT1/2, goat anti-rabbitIgG-HRP and goat anti-mouse IgG-HRP antibodies were purchased from SantaCruz Biotechnology (Santa Cruz, Calif.). Monoclonal mouse anti-βactinwas obtained from Sigma (St. Louis, Mo.). p-AKT (S473), p-AKT (T308),p-S6K (Thr389), p-4E-BP1 (S65), p-4E-BP1 (T37/46), p-S6 (S235/236),p-GSKa/β (Ser21/P), p-ERK1/2 (T202/Y204) antibody were purchased fromCell Signaling Technology (Beverly, Mass.).

B. Cell Culture

Pancreatic cancer cell lines L3.6, PanC-1 and MIA-PaCa2 cells (MIA) weremaintained in DMEM medium (Sigma, St. Louis, Mo.), supplemented with a10% fetal bovine serum (Invitrogen, Carlsbad, Calif.),Penicillin/Streptomycin (1:100, Sigma, St. Louis, Mo.) and 4 mML-glutamine (Sigma, St. Louis, Mo.), in a CO₂ incubator at 37° C. ForWestern blot analysis, cells were reseed in 6-well plates at a densityof 0.5×10⁶ cells/ml with fresh complete culture medium.

C. Cell Viability Assay

Cell viability was measured by the 3-[4m5-dimethylthylthiazol-2-y]-2.5diphenyltetrazolium bromide (MTT) method, as described in Cao et al.(2009) Sci. Signal, 2:RA17. Briefly, the cells were collected and seededin 96-well plate at a density of 2×10⁵ cells/cm². Different seedingdensities were optimized at the beginning of the experiments. Afterovernight incubation cells were exposed to fresh medium containingindicated reagents at 37° C. After incubation for different timeperiods, 20 ul of MTT tetrazolium (Sigma, St. Louis, Mo.) salt dissolvedin Hank's balanced solution at a concentration of 5 mg/ml was added toeach well and incubated in a CO₂ incubator for an additional 4 hours.The medium was subsequently aspirated from each well and 150 ul of DMSO(Sigma, St. Louis, Mo.) was added to dissolve formazan crystals and theabsorbance of each well was obtained using a Dynatech MR5000 platereader at a test wavelength of 490 nm with a reference wavelength of 630nm.

D. Western Blot

As described in Cao et al. (2009) Sci. Signal, 2:RA17, aliquots of 30-40μg of protein from each sample (treated as indicated in the legends)were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE)and transferred onto a polyvinylidene difluoride (PVDF) membrane(Millipore, Bedford, Mass.). After blocking with 10% instant nonfat drymilk for 1 hour, membranes were incubated with specific antibodiesovernight at 4° C. followed by incubation with secondary antibodies(HRP-conjugated anti-rabbit or anti-mouse IgG at the appropriatedilutions) for 60 minutes to 1 hour at room temperature. Antibodybinding was detected with the enhanced chemiluminescence (ECL) detectionsystem (Amersham Biosciences, Piscataway, N.J.).

E. In Vivo Human Pancreatic Tumor Mice Xenograft Model

SCID/Beige/Taconic male mice were inoculated with 2×10⁶ L3.6 pancreaticcells and treated for 4 days post tumor implant with thrice weekly(3×/wk) intraperitoneal (I.P.) injections of paclitaxel (taxol) 3.0mg/ml or gemcitabine (Gem) 10 mg/ml after treatment and were autopsiedwhen near death (as per IRB committee). Mouse survival, tumor volume (incm³), and body weight (in grams) were recorded. Average rate of tumordevelopment was calculated by dividing total tumor volume by totalnumber of days monitored.

Example 2: C6-Ceramide Dramatically Enhances Paclitaxel (Taxol) InducedCell Death in L3.6 Pancreatic Cancer Cells In Vitro

Synergism of paclitaxel and C6-ceramide in killing L3.6 cells in vitrowas tested using an MTT assay in which cell death was reflected asreduced MTT value. C6-ceramide, which alone has limited effects ininducing on L3.6 cell death, dramatically enhanced paclitaxel inducedcell death in a dose dependent manner (FIGS. 1A and 1C). For example,1.5 μg/ml of paclitaxel killed less than 5%, but combination therapycaused more than 90% of L3.6 cell death. Importantly, when L3.6 cellswere cultured in 10% FBS, neither taxol or C6-ceramide alone had anymeaningful effects on L3.6 cell death, whereas a combination of thosetwo agents at a very low concentration caused a significant cell death(FIGS. 1B and 1D). For example, 2.5 μg/ml of C6-ceramide plus 1.5 μg/mlof taxol caused more than 70% cell death of L3.6 cells, while neither ofthese two alone had any measurable effect on L3.6 cell death whencultured in 10% FBS. Thus, synergism between the two agents wasdemonstrated.

Example 3: C6-Ceramide and Taxol Induce Synergistic Anti-Tumor Effect InVivo

As discussed in Example 2, C6-ceramide and taxol exhibited significantsynergism in inducing L3.6 cancer cell death in vitro. Whether possiblesynergistic anti-tumor effects between these two in vivo also occurredwas subsequently determined using the heterotransplanted L3.6 cell modelin SCID mice. As shown in FIGS. 2A and 2B, only the group that receivedcombination taxol (3 mg/ml) and C6-ceramide (10 mg/ml) treatment showedsignificantly smaller tumor volumes and enhanced mouse survival ascompared to the control treatment group and the groups that receivedsingle treatment taxol (3 mg/ml) or C6-ceramide (10 mg/ml). The controland single agent treatments had minimal effects on tumor volume or mousesurvival (FIGS. 2A and 2B). The average rate of tumor development wasreduced to 0.0007 cm³/day in the group receiving both taxol plusC6-ceramide treatment compared to 0.046 cm³/day in the control groups(FIG. 2C). Further, the mean survival was extended to 35.2 days in thegroup receiving combined therapy (taxol/C6-ceramide) compared to 17.8days in control group (FIG. 2D). The body weight of mice that receivedcombined treatment was actually better than the control or individualagent treatment groups indicating relative safety of this strategy (FIG.2E).

Example 4: Synergistic Anti-Tumor Effects of C6-ceramide and GemcitabineIn Vivo and In Vitro

The possible combination effect of C6-ceramide on gemcitabine-inducedcytotoxicity (in vitro) and regression (in vivo) of the L3.6 pancreaticcancer cell was also tested according to the methods described inExamples 2 and 3. As shown in FIGS. 3A-3D, mice that received low dosegemcitabine treatment (5 mg/kg) had limited effect on survival and tumorgrowth. Increasing the dose of gemcitabine to 10 mg/kg caused a moderateanti-tumor effect in the L3.6 cells SCID mice model. For both doses,however, adding C6-ceramide (10 mg/ml) significantly increased theanti-tumor effects significant tumor regression with overall reducedtumor size and significant prolongation of mice survival time in micethat received both C6-ceramide and gemcitabine treatment (FIG. 3E). Asynergistic anti-tumor effect of gemcitabine and C6-ceramide in multiplepancreatic cancer cells in vitro was also determined. As shown in FIGS.3F-3H, either gemcitabine (1.5 μg/ml) or C6-ceramide alone had moderateeffect on cancer cell death, whereas the combination of these two agentscaused a dramatic increase in cell death in pancreatic cancer cell linesL3.6 (FIG. 3F), Panc-1 (FIG. 3G), and MIA cells (FIG. 3H).

Example 5: PI3K/AKT/mTOR Inhibition and AMPK Activation Enhance TaxolInduced Cancer Cell Death

The molecular mechanism involved in C6-ceramide inducedchemosensitization effects was examined by focusing on taxol. Taxolalone is able to strongly induce PI3K/AKT/mTOR and AMPK activation.PI3K/AKT/mTOR inhibition by inhibitors (LY 294002 for PI3K/AKT inhibitorII for AKT and rapamycin for mTOR) and AMPK activation by selectiveactivator (AICAR) enhance Taxol induced cancer cell death (FIGS. 4A-4C).AMPK inhibitors, on the other hand, protect cancer cells fromTaxol-induced cell death (FIG. 4D). C6-ceramide dramatically reducestaxol induced pro-survival PI3K/AKT/mTOR pathway while enhancingpro-apoptotic pathway AMPK/ACC signaling (FIG. 4A). The data in FIG. 6Esuggest that mTORC2 is required for Taxol induced AKT phosphorylation,since SIN1 or mLST8 knockout abolishes AKT phosphorylation.

Example 6: Combination of C6-Ceramide with Gemcitabine or TaxolSynergistically Inactivates Prosurvival (KRAS Pathway) of AKT/mTORC1 andERK In Vitro

The effects of the observed synergistic effects of combination agentsdescribed in Examples 2-4 were extended by analyzing AKT/mTORC1 and ERKactivation, which are two major pro-survival pathways in pancreaticcancer cell lines. As shown in FIG. 5A, gemcitabine itself had no effecton AKT/mTORC1 or ERK activation in the tested time period. C6-ceramideinduced moderate survival of AKT/mTORC1 and ERK activation. However,treatment with a combination of gemcitabine and C6-ceramide caused aprofound inhibition of both AKT/mTORC1 and ERK signaling (FIGS. 5A and5B). AKT/mTORC1 or ERK signaling was also largely inhibited bycombination treatment of taxol plus C6-ceramide (FIGS. 5C and 5D). Thesedata together indicate that C6-ceramide plus gemcitabine or taxol causesin-activation of AKT/mTORC1 and ERK in vitro and may be the keymechanism to explain the observed synergistic anti-cancer effects.

In order to further explore this rational, various inhibitors ofAKT/mTORC1 or ERK signaling pathways were used to assess the signalingpathways shown in FIGS. 5E-5F. The activation of AKT, mTORC1 or ERKsignaling was blocked by various inhibitors. Thus, PI3K/AKT inhibitor LY294002, mTORC1 inhibitor rapamycin, MEK/ERK inhibitor U0126, largelyenhanced taxol and gemcitabine induced L3.6 cell death. BlockingPI3K/AKT/mTORC1 and MEK/ERK signaling by adding both LY 294002 and U0126caused a maximum effect on facilitating taxol or gemcitabine inducedcell death, compared to blocking one signal pathway alone (FIG. 5E,arrow). These data indicate that activation of PI3K/AKT/mTORC1 andMEK/ERK signaling pathways are a major cause of chemoresistance intreatment by anti-cancer agents, such as gemcitabine and taxol.C6-ceramide appears able to reverse the activation of these pathways tothereby potentiate the chemotherapeutic cytotoxic effects on thepancreatic cancer cells. A schematic outline of biological pathwaysaffected by C6-ceramide is shown in FIG. 6.

Taken as a whole, it was found that activation of PI3K/AKT/mTOR pathwayis critical for the resistance against gemcitabine and paclitaxelinduced pancreatic cancer cell death, since inhibitors of PI3K/AKT/mTORlargely sensitized L3.6 pancreatic cancer cells due to gemcitabine andpaclitaxel induced cell death (FIGS. 5E-5F). It was also determined thatadding exogenous cell permeable C6-ceramide caused AKT and downstreammTORC1 inactivation. Interestingly paclitaxel or gemcitabine, which byitself had no effect on PI3K/AKT activation, dramatically enhancedC6-ceramide induced AKT de-phosphorylation or inhibition (FIGS. 5A-5B).However, PI3K/AKT/mTORC1 is not the only signaling pathway that isaffected by exogenous cell permeable C6-ceramide treatment. ERK MAPKsignaling is also inhibited by C6-ceramide treatment. Thus, addingpaclitaxel or gemcitabine enhanced its inhibition on ERK activation(FIGS. 5A-5B). In L3.6 cells, ERK activation is a chemoresistant factor,since U0126, a well characterized MEK/ERK inhibitor, sensitized L3.6cells to gemcitabine and paclitaxel induced cell death (FIG. 5F). Thesedata demonstrate that PI3K/AKT/mTOR inhibition, as well as ERK/MAPKinhibition, by C6-ceramide are likely the key mechanisms for thesynergistic anti-cancer effects described herein.

Example 7: C6-Ceramide Sensitizes KRAS Mutated Pancreatic Cancer Cellsto Cetuximab

Gemcitabine is the first-line chemotherapy for pancreatic cancer, withtaxol as an alternative in experimental models. Chemotherapeutic agentscause side effects and cancer cells often fail to respond adequately dueto acquisition of chemoresistance. Newer targeted therapies have beendeveloped, such as cetuximab, which targets EGF receptors. However, withall of these agents, acquisition of KRAS mutations leads to markedresistance and decreased survival. Oncogenic KRAS mutations occur in 90%of patients with pancreatic cancer, rendering the protein constitutivelyactive with the result that these tumors are highly aggressive and areresistant to chemotherapy.

FIG. 7 demonstrates that C6-ceramide markedly sensitized PANC-1 and MIAPaCa-2 cells (both confirmed to harbor activating KRAS mutations), aswell as L3.6 cells, to cetuximab.

Example 8: Combination of C6-Ceramide with Anti-Cancer AgentsInactivates Pro-Survival Signaling Pathways

One of the most studied scaffold proteins for AKT activation is Gab1.Physical association between p85 and Gab1 is crucial in mediating thePI3K/AKT signaling pathway induced by a variety of stimuli. FIG. 5Fshows that taxol induces Gab1 activation in wildtype but not Gab1knockout MEFs. It is believed that that Gab1 is a key adaptor for taxoland gemcitabine induced PI3K/AKT/mTOR activation and chemoresistanceresponse. To confirm this rationale, wildtype and Gab1 knockout MEFs andGab1 siRNA can be used to test taxol and gemcitabine inducedPI3K/AKT/mTOR and cell death. Interaction between Gab1 and p85 afterTaxol and Gemcitabine treatment can also be tested in L3.6 cells.

In order to determine how anti-cancer agents, such as taxol andgemcitabine, induce mTORC1 activation, experiments can be performed toanalyze AKT and TSC2, two key players for mTORC1 activation. It isbelieved that taxol and gemcitabine induced AKT activation mediates TSC2phosphorylation, which inhibits TSC function as a negative regulator ofmTORC1, leading to phosphorylation at S6K and 4E-BP1. AKT specificinhibitor AKTi and nonspecific inhibitor LY294002 are expected to blockTaxol induced TSC2 phosphorylation (S1462) and mTORC1 activation. AKT1/2deficiency will inhibit TSC2 phosphorylation and mTORC1 activation aswell as enhance Taxol and Gemcitabine induced cancer cell death. It isexpected that high basal level of mTORC1 activation in TSC2 knockoutMEFs would be observed and that taxol and gemcitabine will not inducefurther activation of mTORC1. To further confirm the critical role ofAKT/mTORC1 activation in taxol induced chemoresistance, inhibitors forAKT (AKT inhibitor II) and mTORC1 (rapamycin), with or without taxol andgemcitabine, can be given to pancreatic cancer model SCID mice. It isexpected that those inhibitors enhance Taxol induced cell death in vivo,as indicated by lower mean tumor volume, lower tumor weight, longersurvival rate, and the like in mice with those inhibitors plus taxol.

Based on the observation that taxol-induced AKT phosphorylation at Ser473 is abolished in cells without Sin1 or mLST8, two key components ofmTORC2, it is believed that mTORC2 is more sensitive to Taxol inducedcell death and mTORC2 is important for Taxol to induce chemo-resistance.By using Sin1 and mLST8 deficiency MEF cells, it is expected that themechanisms of phosphorylation of AKT at Ser 473 by taxol will beidentified. It is further expected that taxol-induced AKTphosphorylation at Ser 473 will be abolished in either SIN1 or mLST8knockout MEFs.

In addition, it has been demonstrated herein that taxol alone is able toinduce moderate AMPK activation in pancreatic cell line. For example,AICAR, and AMPK activator, was able to enhance Taxol induced cell death(FIG. 4C), while AMPK inhibitor Compound C inhibits it (FIG. 4D).C6-ceramide dramatically enhances Taxol induced AMPK and its downstreamsignaling ACC phosphorylation in L3.6 cell line (FIG. 4A), which likelyserves as another key mechanism to explain the synergistic effects ofC6-ceramide plus chemotherapy drugs for inducing cancer cell death,since AMPK plays a critical role in tumor-genesis and AMPK activationmediates cancer cell death in a mTOR dependent and independent manner.To further confirm the requirement of AMPK activation in thissynergistic effect, siRNA directed against AMPK can be used. It isexpected that the synergistic effect would be impaired in AMPK knockdowncells. L3.6 cells can be transfected with TSC2 site-specific mutationplasmids to generate TSC2 dominant negative cells and it is expectedthat taxol and ceramide would induce AMPK activation but with lessmTORC1 inhibition in TSC2 dominant cells. In addition, it is expectedthat dominant negative cells would be less sensitive to induced celldeath by a combination of taxol and C6-ceramide.

Taxol directly induces reactive oxygen production (ROS) production andmitochondria stress, ROS is known as a strong AMPK activator. LKB1 iswell-recognized AMPK Kinase. Accordingly, the requirement for ROSproduction in AMPK activation by taxol can be assayed. L3.6 cells withor without antioxidants PDTC or NAC pretreatment can be treated withtaxol, ceramide or taxol plus ceramide and ROS production, LKB1/AMPK/ACCactivation, and L3.6 cell death can be tested. It is expected thatceramide will enhance taxol induced ROS production and LKB1 activation,NAC and PDTC impaired ROS product, LKB1/AMPK/ACC activation, and celldeath induced either by taxol and taxol plus ceramide.

Regarding the relationship between ceramide and KRAS signaling, it hasbeen demonstrated herein that C6-ceramide sensitizes cancer cells tocetuximab to thereby indicate an interaction with the KRAS pathway.Oncogenic Kras stimulates a signaling cascade: Raf/MEK/ERK withsubsequent effects on NF-kB and PI3K/AKT, survival pathways along withthe network effects on jun c, Bcl-2, Bcl-xL. Assessment of in vitroeffects on well-known targets in these pathways can include MTT or XTTcytotoxic assay, caspase 8, and 3, and Tunel assays. The detailedbaseline effects of the KRAS pathway can be evaluated on the anti-tumoreffects of C6-ceramide and selected drug combinations, such aspaclitaxel (taxol) (commonly used in variety of cancers, gemcitabine(currently accepted agent in therapy of pancreatic cancer), andcetuximab (currently used for colorectal and head and neck cancers).

The effect of known RAS inhibitors on the C6-ceramide anti-cancereffects can also be assessed. These can include the farnesyl/transferaseinhibitors: farnesylthiosalicylic acid (Salirasib; Rotblat et al. (2008)Methods Enzymol. 439:467-489, and Tipifarnib (farnesy/proteintransferase inhibitor (R115777; McDonald et al. (2005) Invest. New Drug23:485-487. Similarly, molecular biology approces targeting the KRASsignaling network can be taken by, for example, transfecting cells withpre-let 7a microRNA or Lin 28 siRNA, both of which decrease theexpression of KRAS protein (Kim et al. (2010) Int. J. Radiat. Oncol.Biol Phys. 76:1-5. Alternatively, mutant KRAS can be targeted directlywith an siRNA (Zhu et al. (2006) Cancer Biol Ther. 5:1693-1698).

INCORPORATION BY REFERENCE

All publications, patents and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

Also incorporated by reference in their entirety are any polynucleotideand polypeptide sequences which reference an accession numbercorrelating to an entry in a public database, such as those maintainedby The Institute for Genomic Research (TIGR) on the world wide web attigr.org and/or the National Center for Biotechnology Information (NCBI)on the world wide web at ncbi.nlm.nih.gov.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for treating pancreatic cancer having anactivating KRAS mutation, wherein the activating KRAS mutation is ahuman KRAS polypeptide having a mutation selected from the groupconsisting of G12C, G12A, G12D, G12R, G12S, G12V, G13C, and G13D,comprising administering to a human a pharmaceutically acceptableformulation comprising (a) an effective amount of C6-ceramide; and (b)an effective amount of at least one anti-cancer agent selected from5-fluorouracil, cetuximab and gemcitabine.
 2. The method of claim 1,wherein administering the pharmaceutically acceptable formulation iseffected by parenteral or oral administration.
 3. The method of claim 1,wherein the cancer is affected synergistically with respect toadministering (a) and (b) relative to administering either (a) or (b)alone.
 4. The method of claim 1, wherein the anti-cancer agent comprises5-fluorouracil.
 5. The method of claim 1, wherein the anti-cancer agentcomprises cetuximab.
 6. The method of claim 1, wherein the anti-canceragent comprises gemcitabine.
 7. The method of claim 1, wherein theanti-cancer agent is 5-fluorouracil.
 8. The method of claim 1, whereinthe anti-cancer agent is cetuximab.
 9. The method of claim 1, whereinthe anti-cancer agent is gemcitabine.
 10. The method of claim 2, whereinadministering the pharmaceutically acceptable formulation is effected byparenteral administration; and parenteral administration is selectedfrom intravenous, intraperitoneal, intralymphatic, intralesional, andsubcutaneous.